JP2017500856A - Membrane-permeable peptides for enhanced transfection and compositions and methods using them - Google Patents

Abstract

The present invention is directed to non-natural peptides containing a membrane permeable amino acid sequence and further containing at least one polycationic moiety or peptide sequence. The peptide is suitable for use in delivering cargo to the interior of a cell. Suitable cargoes include nucleic acid molecules (including DNA, RNA, or PNA), polypeptides, or other biologically active molecules. The present invention is further directed to a transfection complex comprising at least one cationic lipid and a non-natural peptide of the present invention non-covalently associated with cargo delivered to the interior of the cell. The invention further relates to methods for preparing and using non-natural peptides for the formation of transfection complexes, and delivery of cargo into the interior of cells in culture, animals or humans. The invention also relates to compositions and kits useful for transfection of cells. [Selection] Figure 9B

Description

  The present invention relates generally to the fields of transfection and cell culture. Specifically, the present invention provides a peptide suitable for use as a cell penetrating peptide, a transfection complex containing the peptide, and its use for intracellular delivery of cargo.

Incorporation by reference All publications, patents, and patent applications mentioned in this specification are intended to indicate that each individual publication, patent, or patent application is specifically and individually incorporated by reference. To the same extent, it is incorporated herein by reference.

  Lipid aggregates such as liposomes are used in the laboratory and clinical research environments to introduce macromolecules such as DNA, RNA, proteins, and small chemicals such as small molecules or pharmaceutically active molecules into cells and tissues. It has been found to be useful as a delivery agent. In particular, lipid aggregates containing a cationic lipid component have been shown to be particularly effective in delivering anionic molecules to cells. For one thing, the effectiveness of cationic lipids and positively charged complexes formed with cationic lipids is believed to be due to increased affinity for cells that have a net negative charge. . For one thing, the net positive charge of lipid aggregates containing cationic lipids allows the aggregates to bind to polyanions such as nucleic acids. Lipid aggregates containing DNA and RNA are known to be effective substances for efficient transfection of target cells.

  The structure of the various types of lipid aggregates varies depending on the composition and method of forming the aggregate. Such aggregates include liposomes, unilamellar vesicles, multilamellar vesicles, micelles and the like having specific sizes on the nanometer to micrometer scale. Methods for making lipid aggregates are generally known in the art. The main drawback of using conventional phospholipid-containing liposomes for delivery is that the liposome composition has a net negative charge and is not attracted to the negatively charged cell surface. By combining cationic lipid compounds with phospholipids, positively charged vesicles, and other types of lipid aggregates, they can bind to negatively charged nucleic acids and can be taken up by target cells, Target cells can be transfected. (Felgner, PL et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7417, Epstein, D. et al., US Pat. No. 4,897,355).

  Methods for incorporating cationic lipids into lipid aggregates are well known in the art. A representative method is described by Felgner et al. (Above), Epstein et al. (Above), Behr et al. (Above), Bangham, A .; et al. (1965) M.M. Mol. Biol. 23: 238-252, Olson, F.M. et al. (1979) Biochim. Biophys. Acta 557: 9-23, Szoka, F .; et al. (1978) Proc. Natl. Acad. Sci. USA 75: 4194-4198, Mayhew, E .; et al. (1984) Biochim. Biophys. Acta 775: 169-175, Kim, S .; et al. (1983) Biochim. Biophys. Acta 728: 339-348, and Fukunaga, M .; et al. (1984) Endocrinol. 115: 757-761. Techniques widely used to prepare lipid aggregates of a size suitable for use as a delivery vehicle include sonication and freeze-thawing in addition to extrusion. For example, Mayer, L. et al. et al. (1986) Biochim. Biophys. See Acta 858: 161-168. Where consistently small (50 nm to 200 nm) and relatively homogeneous aggregates are desired, microfluidization is used (Mayhew, E. (supra)). Cationic lipids have also been used previously to deliver interfering RNA (RNAi) molecules to cells (Yu et al. (2002) PNAS 99: 6047-6052, Harborth et al. (2001) Journal). of Cell Science 114: 4557-4565).

  The use of cationic lipids has become increasingly common since its introduction over 15 years ago. Several cationic lipids are described in the literature, some of which are commercially available. DOTMA (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride) was the first cationic lipid synthesized for nucleic acid transfection purposes. Felgner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987), U.S. Pat. No. 4,897,355). DOTMA may be formulated alone or in combination with DOPE (dioleoylphosphatidylethanolamine) into liposomes, such liposomes can be used to deliver plasmids to some cells. . Other classes of lipids have subsequently been synthesized by various groups. For example, DOGS (5-carboxyspermylglycine dioctadecylamide) is the first polycationic lipid prepared (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989), USA. No. 5,171,678), other polycationic lipids have been prepared since. The lipid DOSPA (2,3-dioleyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-dimethyl-1-propanaminium) has been described as an effective delivery agent (US patent). No. 5,334,761).

  In other examples, cholesterol-based cationic lipids such as DC-Chol (N, N-dimethyl-N-ethylcarboxamide cholesterol) have been prepared and used for transfection (Gao et al. Biochem. Biophys. Res. Comm. 179, 280 (1991)). In another example, 1,4-bis (3-N-oleylamino-propyl) piperazine has been prepared and combined with histone H1 to produce a delivery agent, which is less toxic than other reagents. (Wolf et al. BioTechniques 23, 139 (1997), US Pat. No. 5,744,335). Several reagents are commercially available. Some examples include LIPOFECTIN (R) (DOTMA: DOPE) (Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE (R) (DOSPA: DOPE) (Invitrogen), LIPOFECTAMINE (R) 2000 (InvitroGEN) (Registered trademark), TRANSFECTAM (registered trademark) (DOGS), EFFECTENE (registered trademark), and DC-Chol.

  None of these reagents can be used in common for all cells. This is probably natural given the different types of cell membranes as well as the different barrier compositions that can limit the entry of extracellular material into the cell. Furthermore, the mechanism of delivery of nucleic acids into cells by cationic lipids is not clearly understood. Reagents are less efficient than viral delivery methods and are toxic to cells, but the level of toxicity varies from reagent to reagent.

  However, transfection agents that contain cationic lipids are not effective for all cell types. The effectiveness of transfection of different cells depends on the composition of the particular transfection agent. In general, polycationic lipids are more efficient than monocationic lipids when transfected into eukaryotic cells. In many instances, cationic lipids alone are not effective or only partially effective for transfection.

  The use of lipid aggregates to introduce exogenous compounds into cells (a process known in the art as “transfection”) has become a routine procedure in many laboratories and is widely used. Although adapted for use with cell types and lineages, approximately 60% of cells and cell lines that use this technique routinely in research and clinical settings are considered difficult to transfect. This means that they typically exhibit a transfection efficiency of less than 60%. Cells defined as difficult to transfect include stem cells, progenitor cells, neurons and other cell types derived from neural tissue, primary blood cells ("PBMC"), HUVECs, etc., and have been established However, there are certain cell lines that are difficult to efficiently transfect using commercially available transfection reagents. Examples of cell lines that are difficult to transfect include, among others, PC12, HepG2, 3T3, LNCaP, A549, Jukat, and PC3.

  Over the past decades, several natural peptides that can promote the transfer of substances into cells by passing through the cell membrane. These so-called “membrane permeable peptides” (“MPP”) or “cell permeable peptides” (“CPP”) are used to transport proteins, nucleic acids, polymers, or other functional molecules into cells. ing.

  Penetratin from Antennapedia (Derossi et al., J. Biol. Chem., 269, 10444-10450, 1994) and Tat peptide (Vives et al., J. Biol. Chem., 272, 16010-16017, 1997) Membrane / cell permeable peptides (CPP) have been used to deliver cargo molecules such as peptides, proteins, and oligonucleotides into cells (Fischer et al., Bioconjug. Chem., 12, 825-841). , 2001). The range of applications purely extends from cell biology to biomedical research (Dietz and Bahr, Mol. Cell., Neurosci, 27, 85-131, 2004). Initially, intracellular uptake was thought to occur by direct permeation of the plasma membrane (Prochiantz, Cuff. Opin. Cell Biol., 12, 400-406, 2000). In recent years, there has been increasing evidence that at least some CPPs increase cellular uptake by promoting endocytosis (for discussion see Fotin-Mleckzek et al., Curr. Pharm. Design, 11 , 3613-3628, 2005). In view of these recent results, the specification of a peptide as a CPP / MPP therefore does not necessarily imply a specific intracellular import mechanism, but rather either a covalent or non-covalent bond with the cargo molecule. When bound with, it functions as a peptide that enhances the intracellular uptake of cargo molecules.

  In both research and clinical environments, any cell, especially cells that are considered “difficult to transfect” (ie, standard transfection is ineffective or routinely used in a laboratory environment) Enhanced delivery of cargo and macromolecules into cells by improving the transfection efficiency of any cell that exhibits substantially lower transfection efficiency than a transformed cell line And there is a need for additional reagents that are easy to prepare and utilize the wide variety of cationic lipid-based transfection reagents currently available.

  The present invention provides novel non-natural peptides that have a cell permeation function and are capable of forming transfection complexes with cargo molecules and one or more cationic lipids.

  Disclosed herein are compositions and methods that provide improved efficiency of introducing macromolecules such as nucleic acids into cells in culture or in vivo in tissue. Accordingly, in accordance with certain embodiments, provided herein are complexes comprising a cargo molecule, such as a nucleic acid, a transfection agent, and a non-natural peptide in a non-covalent association. Non-natural peptides contain membrane permeable peptide sequences. In certain embodiments, the complex contains a macromolecule, such as a peptide, protein, or nucleic acid, that is introduced into the cell.

を有し、式中、Ａは、膜透過性ペプチドであり、Ｌは、結合またはリンカーペプチドであり、Ｂは、カチオン性部分またはカチオン性ポリペプチドである。一部の好ましいが非限定的である実施形態では、Ａは、表１に記載されるものから選択されるペプチド配列、またはトランスフェクション効率を強化するその能力の少なくとも一部分を保持するそれらの変異形である。一部の実施形態では、Ａのペプチド配列は、５〜約５０個のアミノ酸であり、Ａは、細胞内への分子の送達を少なくとも５０％以上改善することを特徴とする。 In one aspect of the invention, the non-natural peptide of the invention has the following general structure:

Where A is a membrane permeable peptide, L is a bond or linker peptide, and B is a cationic moiety or cationic polypeptide. In some preferred but non-limiting embodiments, A is a peptide sequence selected from those listed in Table 1, or variants thereof that retain at least a portion of its ability to enhance transfection efficiency. It is. In some embodiments, the peptide sequence of A is from 5 to about 50 amino acids, and A is characterized by improving delivery of the molecule into the cell by at least 50% or more.

  In some embodiments, the peptide sequence of A is from about 5 to about 50 amino acids, and A is characterized by improving delivery of the molecule into the cell by more than 75%.

  In some embodiments, A is at least 75% to any one of the peptides listed in Table 1, or to those variants that retain at least a portion of its ability to enhance transfection efficiency. Identical and A is characterized by improving the delivery of the molecule into the cell by at least 10%. In some embodiments, A is selected from the list consisting of any one of SEQ ID NO: 1 to SEQ ID NO: 68, or variants thereof.

  In one aspect of the invention, the non-natural peptide is selected from any one of the peptides listed in Table 4.

  A further embodiment of the present invention relates to a non-natural peptide as described above with one or more transfection reagents that may comprise one or more cationic lipids, and optionally one or more helper lipids and / or neutral lipids. Targeted are transfection complexes containing in combination.

  In some embodiments, the transfection complex may include cargo delivered to the interior of the cell, or may optionally be administered to an animal or human patient that would benefit from its administration. In some exemplary but non-limiting embodiments, preferred cargo molecules suitable for use in the present invention include nucleic acid molecules such as DNA molecules or RNA molecules. Suitable DNA molecules can include DNA molecules having an expressible nucleic acid sequence, such as an expression vector, or cDNA molecules comprising an open reading frame encoding a protein. Other suitable molecules that can function as suitable cargo molecules in the practice of the present invention include RNA molecules such as mRNA molecules or RNAi molecules.

  Further embodiments of the invention are directed to methods for preparing transfection complexes and methods for using it for delivery of cargo molecules to the interior of a cell. Methods for preparing transfection complexes may include carrying the cargo into the interior of the cell, optionally in the presence of one or more helper lipids and / or one or more neutral lipids. Contacting the cargo molecule with at least one cationic lipid or transfection reagent, as well as a non-natural peptide of the present invention, under conditions that promote the formation of a possible transfection complex.

  Further embodiments of the invention include at least one cargo molecule, at least one cationic lipid or transfection reagent, and a non-natural peptide according to the invention, optionally one or more helper lipids and / or one Transfecting a cell comprising forming a transfection complex having the above neutral lipids and contacting the transfection complex with the cell under conditions that promote transfection of the cell Intended for methods. A still further embodiment of the present invention is a pharmaceutical preparation comprising a cargo or drug delivered to an animal or human subject, at least one cationic lipid or transfection reagent, and a non-natural peptide of the present invention. Optionally in the presence of one or more helper lipids and / or one or more neutral lipids in an animal or human subject in need thereof for the treatment of a physiological condition or disorder Directed to pharmaceutical preparations for forming pharmaceutically active conjugates suitable for the delivery of drugs or biologically active compounds.

  Other embodiments of the invention will be apparent to those skilled in the art from the following drawings and description of the invention, and from the claims.

  The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: Let's go.

FIG. 6 shows a panel of 10 different cancer cell lines expressing GFP transfected with an expression vector encoding green fluorescent protein (GFP) using LIPOFECTAMINE® 3000 in combination with a peptide according to one embodiment. Figure 2 shows two different neural cell lines expressing GFP transfected with an expression vector encoding GFP using LIPOFECTAMINE® 3000 in combination with a peptide according to one embodiment. FIG. 2 shows two different myoblast cell lines expressing GFP transfected with an expression vector encoding GFP using LIPOFECTAMINE® 3000 in combination with a peptide according to one embodiment. FIG. 6 shows a GFP-expressing kidney fibroblast cell line transfected with an expression vector encoding GFP using LIPOFECTAMINE® 3000 in combination with a peptide according to one embodiment. Commercial transfection reagents indicated, FUGENE® HD (first column), LIPOFECTAMINE® 2000 (middle column), and LIPOFECTAMINE® 3000 (last column) in combination with peptides according to one embodiment Column) is used to show a panel of 6 different cell lines expressing GFP transfected with an expression vector encoding GFP. LIPOFECTAMINE® 2000 (white circles), LIPOFECTAMINE® LTX (white squares), and LIPOFECTAMINE® 3000 in combination with peptides according to one embodiment, three different commercially available lipid aggregate formulations. (White triangle) is a graph comparing the relative transfection efficiency of expression vectors encoding GFP transfected into cultured HeLa cells. LIPOFECTAMINE® 2000 (open circle), LIPOFECTAMINE® LTX (open square), and LIPOFECTAMINE® 3000 in combination with peptides according to one embodiment, with increasing doses of three different commercially available lipid aggregate formulations. It is the graph which compared the intensity | strength of the expression of GFP in the HeLa cell which transfected the expression vector which codes (GFP) using (white triangle). Western blot comparing GST-STAT fusion protein (upper panel) in HepG2 cells transfected with an expression vector encoding GST-STAT fusion protein using the following commercially available lipid aggregate formulation: LIPOFECTAMINE® ) 2000 (first lane), LIPOFECTAMINE® 3000 (second lane), FUGENE® HD (third lane), and X-TREMEGENE ™ combined with peptides according to one embodiment HP (last lane). The lower panel shows a western blot of endogenous β-actin to confirm equal cytoplasmic extract loading in each lane. Increasing doses of GFP expression vector using either 0.1-0.6 μl per well of LIPOFECTAMINE® 2000 (white triangles) or LIPOFECTAMINE® 3000 in combination with peptides according to one embodiment A graph comparing the relative transfection efficiencies of H9 human embryonic stem cell lines (37,500 cells per well in a 96-well plate) transfected with (50 μg left panel, 100 μg center panel, 200 μg right panel). is there. Using either 200 μl of LIPOFECTAMINE® 2000 (left panel, showing 18% H9 cell transfection efficiency) or LIPOFECTAMINE® 3000 (right panel) in combination with a peptide according to one embodiment. FIG. 5 is a representative fluorescence image of GFP expression in H9 cells cultured in 96-well plates transfected at 100 μg / well. Measured as mean orange fluorescent protein (OFP) intensity (bar graph, upper panel) in modified U2OS cells using the approximate LIPOFECTAMINE® 2000 shown or LIPOFECTAMINE® 3000 in combination with a peptide according to one embodiment. The efficiency of genome modification of U2OS cells using a commercially available system and a representative fluorescence image of OFP (lower panel) is shown. A commercial system, measured as OFP intensity (bar graph, top panel) in modified HepG2 cells using the approximate LIPOFECTAMINE® 2000 shown or LIPOFECTAMINE® 3000 in combination with a peptide according to one embodiment. The genome modification efficiency of the HepG2 cells used and representative fluorescence images of the OFP (lower panel) are shown. FIG. 6 shows the cleavage efficiency of TALEN and CRISPR targeting the AAVS1 locus in U2OS cells using either LIPOFECTAMINE® 2000 or LIPOFECTAMINE® 3000 in combination with a peptide according to one embodiment. FIG. 5 shows the cleavage efficiency of TALEN and CRISPR targeting the AAVS1 locus in HepG2 cells using either LIPOFECTAMINE® 2000 or LIPOFECTAMINE® 3000 in combination with a peptide according to one embodiment. LIPOFECTAMINE® 3000 alone (LF3K), LIPOFECTAMINE® 2000 (LF2K), peptide according to one embodiment (peptide 1), or LIPOFECTAMINE in combination with a peptide according to one embodiment (in units of μl) Relative transfection efficiency of HeLa cells transfected with GFP expression vector (upper graph, GFP as a percentage of single cells) or relative per cell using 3000 (LF3K + peptide 1) Is a bar graph representing the expression level (lower graph, average FL-1 of single cells only); 9B and 9C represent peptide maps of various peptides or peptide fragments used in the experiments in HepG2 cells shown in FIGS. 9B and 9C, where peptide A is MPP peptide alone, peptide B is linker peptide alone, peptide C is a cationic peptide alone, peptide D is a linker peptide fused to a cationic peptide, and peptide E is a full-length peptide in which peptide A is fused to peptide D. For detecting GFP expression in HepG2 cells transfected with an expression vector encoding GFP using LIPOFECTAMINE® 3000 in the presence of the indicated peptide or peptide combination (shown in FIG. 9A) Represents a series of fluorescent images. Mean fluorescence per cell in HepG2 transfected with an expression vector encoding GFP using LIPOFECTAMINE® 3000 in the presence of one of the indicated peptides A to E or a combination of possible peptides Two bar graphs representing (upper graph) and transfection efficiency (% GFP + cells) are represented (FIG. 9A). 10B and 10C represent peptide maps of various peptides or peptide fragments used in the experiments in A549 cells shown in FIGS. 10B and 10C, where peptide A is MPP peptide alone, peptide B is linker peptide alone, peptide C is a cationic peptide alone, peptide D is a linker peptide fused to a cationic peptide, and peptide E is a full-length peptide in which peptide A is fused to peptide D. Detecting expression of GFP in cultured A549 cells transfected with an expression vector encoding GFP transfected with LIPOFECTAMINE® 3000 in the presence of the indicated peptide or peptide combination (shown in FIG. 10A). Represents a series of fluorescent images. A549 cells transfected with an expression vector encoding GFP using LIPOFECTAMINE® 3000 in the presence of one of the indicated peptides A to E or a combination of the indicated peptides (shown in FIG. 10A) 2 bar graphs showing the mean fluorescence per cell in (top graph) and transfection efficiency (% GFP + cells). 11B and 11C represent peptide maps of various peptides or peptide fragments used for experiments in MDA-MB-231 cells shown in FIGS. 11B and 11C, where peptide A is MPP peptide alone and peptide B is linker peptide alone. Peptide C is a cationic peptide alone, peptide D is a linker peptide fused to a cationic peptide, and peptide E is a full-length peptide in which peptide A is fused to peptide D. Expression of GFP in cultured MDA-MB-231 cells transfected with an expression vector encoding GFP transfected with LIPOFECTAMINE® 3000 in the presence of the indicated peptide or peptide combination (shown in FIG. 11A) Represents a series of fluorescent images to detect. MDA-transfected with an expression vector encoding GFP using LIPOFECTAMINE® 3000 in the presence of one of the indicated peptides A to E or a combination of possible peptides (shown in FIG. 11A) Two bar graphs showing mean fluorescence per cell (upper graph) and transfection efficiency (% GFP + cells) in MB-231.

  The present invention provides improved reagents and compositions that are suitable for transfection of cells. Specifically, the present invention provides compositions and reagents that enhance the transfection efficiency of any cell, including cell types that are typically considered difficult to transfect. The compositions and reagents of the present invention are typically transfections of such when used according to the methods described herein and according to general knowledge and expertise within the skill of the artisan. Efficiency up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80% , Up to 85%, up to 90%, up to 95%, up to 100%, or more than 100%. The present invention accomplishes this by providing a novel peptide comprising a cell / membrane permeable peptide sequence used in combination with one or more transfection lipids, as described in more detail below. To do.

Definitions Terms used throughout this specification generally have their ordinary meanings in the art, within the context of this invention, and in the specific context where each term is used. In describing various embodiments of the present invention and methods of making and using the same, certain terms are discussed below or elsewhere in this specification to provide further guidance to the practitioner. The It is understood that the same concept may be expressed in more than one way. As a result, alternative terms and synonyms may be used for any one or more of the terms discussed herein, with respect to whether the terms are described or discussed in greater detail herein. Does not have any special significance. Synonyms for certain terms may be provided. The listing of one or more synonyms does not exclude the use of other synonyms. The use of examples in any part of this specification, including examples of any terms described herein, is merely exemplary and in no way limits the scope and meaning of the present invention and any exemplified terms. It is not limited.

  When the term “introduction” is used in the context of introducing a macromolecule into a cell culture, the purpose of introducing the macromolecule allows the macromolecule to be transferred from the extracellular compartment of the cultured cell to the cytoplasmic compartment. To provide a macromolecule or compound to the culture medium.

  The term “introducing” a macromolecule or compound into at least one cell refers to providing the macromolecule or compound to the cell such that the macromolecule or compound is internalized into the cell. For example, the macromolecule or compound may be introduced into the cell using transfection, transformation, injection, and / or liposome introduction, and introduced into the cell using other methods known to those skilled in the art. May be. Preferably, the macromolecule or compound is introduced into the cell by liposome introduction. The macromolecule is preferably a protein, peptide, polypeptide or nucleic acid. The macromolecule can be a protein. Alternatively, the macromolecule may be a peptide. Alternatively, the macromolecule may be a polypeptide. The macromolecule may also be a nucleic acid.

  The term “cargo” as used herein in the context of delivering cargo to the interior of a cell, such as by transfection, is generally either in culture in a laboratory or in tissue in an animal or human. It refers to any substance that is carried into the cell. The cargo may be a macromolecule such as a nucleic acid, protein or peptide, or a drug or other small organic molecule, depending on the application.

  The term “macromolecule” as used herein encompasses biomolecules. In one embodiment, the term macromolecule refers to a nucleic acid. In a preferred embodiment, the term macromolecule refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In some embodiments, the term macromolecule refers to DNA. The DNA can be either linear DNA or circular DNA, for example, DNA in the form of a circular plasmid, episome, or expression vector. In certain preferred but non-limiting embodiments, the term macromolecule comprises at least one operably linked to one or more nucleic acid sequences required for transcription of mRNA from an expressible nucleic acid sequence. Refers to complementary DNA (cDNA) having an expressible nucleic acid sequence, including an open reading frame. The macromolecule may be charged or uncharged. A DNA molecule is an example of a charged macromolecule. In some cases, the term “macromolecule” as used herein may be used interchangeably with the terms “expressible nucleic acid” and “expression vector”. In other embodiments, the term “macromolecule” refers to an RNA molecule. An RNA molecule can be any type of RNA, including but not limited to mRNA, siRNA, miRNA, antisense RNA, ribozyme, or any other type or species of RNA well known to those skilled in the art without limitation. It can be RNA and these are envisaged to be delivered inside the cell.

  The term “transfection” is used herein to mean delivering a nucleic acid, protein, or other macromolecule to a target cell, so that the nucleic acid, protein, or other macromolecule is Expressed in or have biological function.

  As used herein, the term “expressible nucleic acid” includes both DNA and RNA, regardless of molecular weight, and the term “expression” refers to any functional presence of the nucleic acid in a cell. Means manifestation and includes both transient expression and stable expression without limitation. Functional aspects include inhibition of expression by oligonucleotide or protein delivery.

  The term “nucleic acid expression” and their equivalents refer to nucleic acid replication in cells, transcription of DNA into messenger RNA, translation of RNA into protein, post-transcriptional modification of protein, and / or protein into cell. Refers to transportation, or variations or combinations thereof.

  The term “cell” as used herein refers to and includes all types of eukaryotic and prokaryotic cells. In preferred embodiments, the term refers to eukaryotic cells, particularly cells grown in culture or found in animal or human tissue. In a preferred embodiment, the cell refers to a mammalian cell. In certain exemplary but non-limiting embodiments, the term “cell” is meant to refer to any cell and cell line that is routinely used in research and clinical settings. Can include, but is not limited to, immortalized cell lines, transformed cell lines, or primary cells.

  The phrase “difficult to transfect” or similar variations of this phrase, when used in the context of transfection procedures and reagents, includes, for example, cationic lipids (for example, LIPOFECTAMINE® 2000, Standard commercially available products such as, but not limited to, LIPOFECTAMINE (registered trademark) LTX, LIPOFECTAMINE (registered trademark), LIPOFECTIN (registered trademark), FUGENE (registered trademark) HD, X-TREMEGENE (registered trademark) HP, etc. A relative term that generally refers to any cell or cell line that typically exhibits a transfection efficiency of less than 60% when transfected using a transfection reagent. Cells typically considered “difficult to transfect” include primary cells such as stem cells, progenitor cells, neurons and other cell types derived from neural tissue, primary blood cells (“PBMC”), HUVEC and the like, as well as certain cell lines that have been established but are difficult to efficiently transfect using commercially available transfection reagents. Examples of cells that are difficult to transfect include PC12, HepG2, 3T3, LNCaP, A549, Jurkat, primary cells, H9 embryonic stem cells, cultured embryonic stem cells, culture-induced pluripotent stem cells (iPS cells), K-562, L6, L929, MCF-7, RAW 264.7, HT29, U937, Vero, HCT116, C6, C2C12, HL60, THP1, BHK, PC3, P19, SH-SY5Y, U2OS, HUH7, and PC3 Examples include, but are not limited to: Reference herein to specific cell types and cell lines considered within the skill of the art in no way is meant to limit the scope of the invention to only those cell lines or approximate derivatives thereof. Simply meant to illustrate the large number of cell lines and types widely used in laboratory environments that exhibit transfection efficiency of less than 60% using commonly available cationic lipid-based transfection reagents, These will help to increase the relative transfection efficiency by at least 5% or more with the novel compositions and formulations described herein.

  “Cell culture” or “culture” means the maintenance of cells in an artificial in vitro environment.

  “Recombinant protein” refers to a protein encoded by a nucleic acid introduced into a host cell. The host cell expresses the nucleic acid. The term “expressing a nucleic acid” is synonymous with “expressing a protein from RNA encoded by the nucleic acid”. As used herein, “protein” generally refers to any natural or synthetic amino acid polymer, such as peptides, polypeptides, proteins, lipoproteins, glycoproteins, and the like.

  As used herein, the term “polypeptide” generally refers to length or post-transcriptional modifications (eg, cleavage, phosphorylation, glycosylation, acetylation, methylation, isomerization, reduction, farnesylation). Etc.) refers to natural, recombinant, or synthetic amino acid polymers that are covalently linked to each other by sequential peptide bonds. “Large” polypeptides are typically referred to in the art as “proteins,” but the terms “polypeptide” and “protein” are often used interchangeably. In general, the first amino acid residue or group of amino acid residues in a polypeptide is referred to as the “amino terminus” or “N-terminus” of the polypeptide. Similarly, the last amino acid residue or group of amino acid residues in a polypeptide is referred to as the “carboxy terminus” or “C terminus”.

  As used herein, the term “peptide” refers to short peptides (typically less than 100 amino acids), polypeptides (typically more than 100 amino acids), and proteins (one or more (Contains a polypeptide chain) is intended to be a broad term, and peptides of the invention typically have more than two amino acids, and preferred peptides include four Has more amino acids.

  As used herein in the context of the polypeptides described herein, terms such as “variant (s)” generally refer to a polypeptide (s) structurally similar to a reference polypeptide. This is a difference in amino acid sequence between this polypeptide and a reference polypeptide (e.g., at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 85%, Or having at least 95% sequence identity) and / or the presence or absence of one or more biochemical modifications (eg, post-transcriptional modifications, substitutions, addition of adducts, etc.). Although the general subset of activities of a particular variant can be similar, the structural differences that occur between variants can result in at least a portion of their activities not overlapping. A “variant” refers to the addition, deletion, substitution, and covalent attachment of one or more consecutive amino acids in a sequence to a polypeptide molecule at one or more positions in the polypeptide sequence. It may refer to a polypeptide molecule that has been altered, including modifications. Thus, in some cases, the terms “variant” and “isoform” may be used interchangeably. Illustrative examples of such variants include, by way of example only, polys in which replacement of hydrogen groups by alkyl, acyl, thiol, amide, or other such functional group has occurred at one or more amino acid residues. Peptides may be mentioned. Variants have “conservative” changes (eg, replacement of a non-polar amino acid residue with another non-polar amino acid residue) where the substituted amino acid may have similar structural and / or chemical properties There is. Variants may also have “nonconservative” changes (eg, replacement of polar amino acid residues with nonpolar or charged amino acid residues). Variants may also include similar minor variations in amino acid sequences, including but not limited to deletions, truncations, insertions, or combinations thereof. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without negating or otherwise substantially affecting biological activity is Are widely available in the art. Further guidance can be found using computer programs well known in the art, such as DNASTAR software. In general, and in the context of the present invention, a variant is typically a cargo molecule that is a subset of at least one biological function associated with a known membrane-permeable peptide, for example, a nucleic acid molecule or the like. It will retain the ability to facilitate translocation across the cell membrane into its cytoplasmic compartment.

  As used herein, the term “amino acid” generally refers to naturally occurring or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Natural amino acids are those encoded by the genetic code, as well as later modified amino acids such as hydroxyproline, carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, i.e., a hydrogen, a carboxyl group, and an alpha carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, methionine, and methylsulfonium. . Such analogs have modified R groups (eg, norleucine or norvaline) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The term “amino acid” may refer to amino acids or their derivatives (eg, amino acid analogs) and their D and L forms. Examples of such amino acids include glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L- Examples include leucine, L-glutamine, L-arginine, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, N-acetylcysteine. It is done.

  “Kit” refers to a kit for transfection, delivery of DNA, RNAi, or other cargo (eg, a protein or anionic molecule), or expression or knockdown of a protein, including the reagents of the present invention. One or more or mixtures thereof are included. The kit can include one or more of the non-natural peptides described herein, optionally with one or more cationic lipids or transfection reagents. In some embodiments, the peptide and lipid reagents can be provided in a single formulation. In other embodiments, the lipid and peptide may be provided separately along with instructions for the user to match the reagents at the time of use. Such a kit may include a segmented holding means for tightly constraining and receiving one or more containment means such as vials, test tubes. Each such containment means contains a component or mixture of components required to effect transfection. Such kits are, for example, nucleic acids (preferably one or more expression vectors, DNA molecules, RNA molecules, or RNAi molecules), cells, one or more compounds of the invention, lipid aggregate-forming compounds, transfection enhancers. Optionally, one or more components selected from any cargo molecule, such as a biologically active substance, and the like.

  The media, methods, kits, and compositions of the present invention include either cell monolayers or suspension cultures, transfection and culture, and proteins in cells in monolayers or suspension cultures. It is suitable for expression. Preferably, the media, methods, kits, and compositions of the invention are suitable for cell culture, transfection, and culture, and for expression of protein products in cells in suspension culture. is there.

  “Culture vessel” means any vessel, eg, a vessel made of glass, plastic, or metal that can provide a sterile environment for culturing cells.

  The term “combine” refers to mixing or mixing the ingredients.

  The term “vector”, as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA that may be ligated with additional DNA segments. Another type of vector is a phage vector. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell's genome upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, certain vectors can direct expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” or simply “expression vectors”. In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. Certain vectors used in the practice of the invention described herein can be well-known vectors used in the art, such as, for example, pCDNA3.3, or modified forms thereof. Non-limiting examples of types of modifications to vectors that may be suitable in the practice of the present invention include one or more enhancers, one or more promoters, one or more ribosome binding sites, one or more origins of replication, etc. Modifications such as the addition of the above-mentioned modifications can be mentioned, but are not limited thereto. In certain preferred but non-limiting embodiments, the expression vector used in the practice of the invention is one or more selected to enhance expression of the protein of interest in the transient expression system. Can be mentioned. The selected enhancer element can be located 5 ′ or 3 ′ of the expressible nucleic acid sequence used to express the protein of interest.

  As used herein, the phrase “expression vector containing an expressible nucleic acid” generally refers to the desired protein of interest (the protein of interest is selected by the user of the present invention). An expressible nucleic acid sequence having at least one open reading frame, in addition to one or more nucleic acid sequences or elements required to assist its expression in a cell or cell-free expression system. It refers to a vector as defined above that can. Such additional nucleic acid sequences or elements that may be present in an expression vector as defined herein include one or more promoter sequences, one or more enhancer elements, one or more ribosome binding sites, one or more Transcription start sequences, one or more origins of replication, or one or more selectable markers. Various nucleic acids or elements suitable for this purpose are well known to those of skill in the art, and the selection of one or more for use in the practice of the present invention is well within the skill of the artisan.

  The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to any nucleic acid, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In preferred embodiments, “nucleic acid” refers to DNA, including genomic DNA, complementary DNA (cDNA), and oligonucleotides including oligo DNA. In certain preferred but non-limiting embodiments, “nucleic acid” refers to genomic DNA and / or cDNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or synthetic reactions. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be made before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide elements. A polynucleotide may include modification (s) made after synthesis, such as conjugation to a label. Other types of modifications include, for example, “caps”, substitution of one or more natural nucleotides with analogs, internucleotide modifications, eg, by uncharged bonds (eg, methylphosphonate, Esters, phosphoamidates, carbamates, etc.) and those by charge bonds (eg phosphorothioates, phosphorodithioates, etc.), eg proteins etc. (eg nucleases, toxins, antibodies, signal peptides, poly-L-lysine etc.) Those containing pendant moieties, those using intercalators (eg, acridine, psoralen, etc.), those containing chelating substances (eg, metals, radioactive metals, metal oxides, etc.), those containing alkylating substances, modified bonds (eg, α Anomeric nucleic acids, etc.) and polynucleotide (s) It includes unmodified forms of. In addition, any of the hydroxyl groups normally present in sugars can be replaced by, for example, phosphonate groups, phosphate groups, protected with standard protecting groups, or further linked to additional nucleotides. It may be activated to prepare or may be joined to a solid or semi-solid support. The 5 ′ and 3 ′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides may also include, for example, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro-, or 2′-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, Ribose or deoxy, commonly known in the art, including epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugar, furanose sugar, cedoheptulose, acyclic analogs, and basic nucleoside analogs such as methyl riboside It may contain analog forms of ribose sugars. One or more phosphodiester bonds may be replaced with alternative linking groups. Among these alternative linking groups are phosphoric acid P (O) S (“thioate”), P (S) S (“dithioate”), (O) NR2 (“amidate”), P (O) R. , P (O) OR ′, CO, or CH 2 (“formacetal”), including but not limited to, wherein R or R ′ is independently H, Or substituted or unsubstituted alkyl (1-20 C) (optionally including an ether (--O--) bond), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. All bonds of a polynucleotide need not be identical. The above applies to all polynucleotides referred to herein, including RNA and DNA.

  As used herein, the term “RNA interference” or “RNAi” generally refers to a sequence-specific post-transcriptional gene silencing process. RNAi is a process in which specific mRNA is degraded into short RNAs. To mediate RNAi, double stranded RNA (dsRNA) having substantial sequence identity to the target mRNA is introduced into the cell. The target mRNA is then degraded in the cell, resulting in a reduced level of that mRNA and the protein it encodes.

  As used herein, the term “RNAi construct” generally refers to small interfering RNA (siRNA), hairpin RNA, and other RNA species that can be cleaved in vivo to form siRNA. The term also encompasses expression vectors that can yield transcripts that form dsRNA or hairpin RNA in cells and / or that can generate siRNA in vivo. The term “RNAi expression vector” refers to a replicable nucleic acid construct, which is used to express (transcribe) RNA that produces siRNA duplexes in a host cell in which the construct is expressed.

  As used herein, the term “short interfering RNA” or “siRNA” generally refers to a short (approximately 19 to about 25 nucleotides in length) of a defined nucleotide sequence capable of mediating RNAi. Refers to a double-stranded RNA molecule.

  As used herein, terms such as “complex formation reaction”, “complex formation medium” and the like generally refer to a physiologically acceptable culture in which nucleic acids are complexed into a transfection reagent formulation. Refers to medium or reaction. Typically, a nucleic acid to be introduced into a cell for protein expression is first complexed with a suitable transfection reagent (eg, a cationic lipid formulation) to form a lipid / nucleic acid complex. It becomes a body or an aggregate.

  A drug refers to any therapeutic or prophylactic agent other than food that is used to prevent, diagnose, alleviate, treat, or cure a disease in a human or animal.

  A variety of techniques and reagents are available for introducing macromolecules into target cells in a process known as “transfection”. Widely used reagents include, for example, calcium phosphate, DEAE-dextran, and lipids. For examples of detailed protocols for using these types of reagents, see numerous reference documents, eg, Current Protocols in Molecular Biology, Chapter 9, Ausubel, et al. Eds. John Wiley and Sons, 1998 is available. Additional methods for transfection of cells are known in the art, including electroporation (electrotransfer of genes), ultrasonic drilling, optical transfection, protoplast fusion, imperfection ( There may be mentioned implication, magnetofection, or viral transduction.

A “reagent for introduction of a macromolecule” or “transfection reagent” into a cell is any material, formulation, or composition known to those of skill in the art that facilitates entry of the macromolecule into the cell. See, for example, US Pat. No. 5,279,833. In some embodiments, the reagent can be a “transfection reagent” and can be any compound and / or composition that increases the uptake of one or more nucleic acids into one or more target cells. Various transfection reagents are known to those skilled in the art. Suitable transfection reagents include cationic polymers such as polyethyleneimine (PEI), polymers of positively charged amino acids such as polylysine and polyarginine, positively charged and split dendrimers, cationic β-cyclodextrin One or more compounds and / or compositions, including but not limited to polymers (CD polymers), DEAE-dextran, and the like. In some embodiments, reagents for introduction of macromolecules into cells can include one or more lipids that can be cationic lipids and / or neutral lipids. Preferred lipids include N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylcholine (DOPE), 1,2-bis (ole). Oiloxy) -3- (4′-trimethylammonio) propane (DOTAP), 1,2-dioleoyl-3- (4′-trimethylammonio) butanoyl-sn-glycerol (DOTB), 1,2-dioleoyl- 3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl (4′-trimethylammonio) butanoate (ChoTB), cetyltrimethylammonium bromide (CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide ( DORI), 1,2-Geolay Oxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), O, O′-didodecyl-N- [p (2- Trimethylammonioethyloxy) benzoyl] -N, N, N-trimethylammonium chloride, spermine conjugated to one or more lipids (eg, 5-carboxyspermylglycine dioctadecylamide (DOGS), N, N ^I , N ^{II, N III} - tetramethyl ^{-N, N I, N II,} N III - tetra palmityl spermine (TM-TPS), and dipalmitoyl Rufasu phosphatidyl ethanolamine 5-carboxyfluorescein spelling mill amine de (DPPES)), lipoic Polylysine (DOP Polylysine), TRIS (tris (hydroxymethyl) aminomethane, tromethamine), and / or peptides such as trilysyl-alanyl-TRIS mono, di, and tripalmitate (3β- [N- -(N ', N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol), N- (α-trimethylammonioacetyl) -didodecyl-D-glutamate chloride (TMAG), dimethyldioctadecylammonium bromide (DDAB) ), 2,3-dioleyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), and combinations thereof, Not limited to

  One skilled in the art understands that certain combinations of the above lipids have been shown to be particularly suitable for introduction of nucleic acids into cells, eg, 3: 1 (by weight of DOSPA and DOPE / Weight) is a combination of Life Technologies Corporation, Carlsbad, Calif. Available under the trade name LIPOFECTAMINE ™ and a 1: 1 (weight / weight) combination of DOTMA and DOPE is available from Life Technologies Corporation, Carlsbad, Calif. Available under the trade name LIPOFECTIN® and a 1: 1 (M / M) combination of DMRIE and cholesterol is available from Life Technologies Corporation, Carlsbad, Calif. Available under the trade name DMRIE-C Reagent and a 1: 1.5 (M / M) combination of TM-TPS and DOPE is available from Life Technologies Corporation, Carlsbad, Calif. Available under the trade name CELLFECTIN® and a 1: 2.5 (weight / weight) combination of DDAB and DOPE is available from Life Technologies Corporation, Carlsbad, Calif. Is available under the trade name LIPFECTACE®. In addition to the lipid combinations described above, other formulations are known to those skilled in the art that include lipids in admixture with other compounds, particularly in admixtures with peptides and proteins containing nuclear localization sequences. . For example, see International Application No. PCT / US99 / 26825, published as International Publication No. WO 00/27795, both of which are incorporated herein by reference.

  Lipid aggregates such as liposomes have been found useful as drugs for delivery of macromolecules into cells. In particular, lipid aggregates comprising one or more cationic lipids have been shown to be very efficient in delivering anionic macromolecules (eg, nucleic acids) into cells. One widely used cationic lipid is N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA). Liposomes containing DOTMA alone or as a 1: 1 mixture with dioleoylphosphatidylethanolamine (DOPE) have been used to introduce acid into cells. A 1: 1 mixture of DOTMA: DOPE is available from Life Technologies Corporation, Carlsbad, Calif. Commercially available under the trade name LIPOFECTIN ™. Another cationic lipid that has been used to introduce nucleic acids into cells is 1,2-bis (oleoyl-oxy) -3--3- (trimethylammonia) propane (DOTAP). DOTAP differs from DOTMA in that the oleoyl moiety is bound to the propylamine backbone by an ether bond in DOTAP, while it is bound by an ester bond in DOTMA. DOTAP is seen to be more easily degraded by target cells. A structurally related group of compounds in which one of the methyl groups of the trimethylammonium moiety is replaced with a hydroxyl group is structurally similar to the Rosenthal inhibitor (RI) of phospholipase A (Rosenthal, et al., (1960) J. Biol. Chem. 233: 2202-2206.). The RI has a stearoyl ester attached to a propylamine core. The dioleoyl analog of RI is generally abbreviated as DOR1-ether and DOR1-ester, depending on the linkage between the lipid moiety and the propylamine core. The hydroxyl group of the hydroxyethyl moiety may be further derivatized with carboxyspermine, for example by esterification.

  Another class of compounds that have been used to introduce macromolecules into cells contains a carboxyspermine moiety bound to a lipid (Behr, et al., (1989) Proceedings of the National Academy of Sciences, USA 86. : 6982-6986 and European Patent 0 394 111). Examples of this type of compound include dipalmitoyl phosphatidylethanolamine 5-carboxyspermylamide (DPPES) and 5-carboxyspermylglycine dioctadecylamide (DOGS). DOGS is available from Promega, Madison, Wis. Commercially available under the trade name TRANSFECTAM ™.

  Cationic derivatives of cholesterol (3β- [N- (N ′, N′-dimethylaminoethane) -carbamoyl] cholesterol, DC-Chol) have been synthesized and formulated into liposomes along with DOPE (Gao, et al (1991) BBRC 179 (1): 280-285.), Which has been used to introduce DNA into cells. It has been reported that liposomes formulated in this way efficiently introduce DNA into cells at low cytotoxic levels. Lipopolylysine (Zhou, et al., (1991) BBA 1065: 8-14) formed by conjugating polylysine to DOPE is effective in introducing nucleic acids into cells in the presence of serum. Has been reported.

  Other types of cationic lipids that have been used to introduce nucleic acids into cells include US Pat. Nos. 5,674,908 and 5,834,439, and International Publication No. WO 00/27795. Highly loaded polycationic ammonium, sulfonium, and phosphonium lipids such as those described in published International Application No. PCT / US99 / 26825 may be mentioned. One particularly preferred but non-limiting transfection reagent for macromolecule delivery according to the present invention is LIPOFECTAMINE 2000 ™, which is available from Life technologies (International Publication No. WO 00/27795). No. PCT / US99 / 26825, published as US No. PCT / US99 / 26825). Another preferred but non-limiting transfection reagent suitable for delivery of macromolecules to cells is EXPIFECTAMINE ™. Other suitable transfection reagents include LIOFECTAMINE ™ RNAiMAX, LIPOFECTAMINE ™ LTX, OLIGOFECTAMINE ™, Cellfectin ™, INVIVOFECTAMINE ™, US by INVIFECTAMINE ™ et al. 2.0, and Yanga Any of the lipid reagents or formulations disclosed in patent application 2012/0136073 (incorporated herein by reference) may be mentioned. A variety of other transfection reagents are known to those of skill in the art and can be evaluated for their suitability for the transient transfection systems and methods described herein.

  The present invention provides improved reagents and compositions that are suitable for transfection of cells. Specifically, the present invention provides compositions and reagents that enhance the transfection efficiency of any cell, including cell types that are typically considered difficult to transfect. The compositions and reagents of the present invention are typically used to transfer such cells when used according to the methods described herein and according to general knowledge and expertise within the skill of the artisan. Up to 10%, up to 15%, 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, Up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, or more than 100%. The invention relates to cells in culture or in vivo cells or tissues, particularly, but not limited to, cells that are considered “difficult to transfect” or as described in more detail below. A cell / membrane permeable peptide used in combination with one or more transfection lipids to deliver a cargo molecule, specifically but not limited to a nucleic acid molecule, such as a DNA or RNA molecule, in the cytoplasmic compartment This is achieved by providing a novel peptide comprising the sequence.

Membrane / cell permeable peptide The present invention is directed to non-natural synthetic peptides used in combination with transfection reagents, preferably lipid-based transfection reagents, particularly cationic lipid-based transfections. Reagents can include, but are not limited to, any other suitable cargo such as a cargo molecule, such as a nucleic acid or one that would be readily apparent to one of skill in the art, by including this peptide in the transfection complex. By enhancing the transport of molecules across the cell membrane, the transfection efficiency of the cells is partially improved by allowing the cargo molecules to be delivered to the cytoplasmic compartment of the cells in culture or in vivo tissue.

  Ideally, a non-natural peptide of the invention is a multi-component complex having a lipid aggregate composition and a cargo molecule such that the complex enhances delivery of the cargo molecule to the cytoplasmic compartment of a cell or tissue. Will be used to form the body.

  In one aspect of the invention, a non-natural peptide is contacted with at least one transfection reagent and at least one cargo molecule to form a transfection complex comprising the transfection reagent, the cargo and the peptide; Improve the transfection efficiency of the complex (measured as an improvement in cargo transport into the interior of the cell in cell fluid or in vivo tissue) compared to the same transfection complex without the non-natural peptide It is characterized by that.

  The choice of constructing an optimal transfection reagent for use in the present invention is the identity and nature of the cargo to be delivered, the identity and characteristics of the cells to be transfected (the transfection attenuation is the isolated cells in the culture medium). Depending on the identity of the non-natural peptide. All of these features are well known to those skilled in the art, and the choice of the optimal transfection reagent in a particular situation for a particular application and the means of determining what constitutes the optimal concentration and formulation of the components is unnecessary experimentation. It will be readily apparent to those skilled in the art without having to do so and without departing from the spirit and scope of the invention.

  In certain preferred but non-limiting embodiments, the transfection reagent selected for use in forming the transfection complex according to embodiments described herein is a cationic lipid, particularly a lipid. It can be a cationic lipid capable of forming an aggregate.

Ａは、膜透過性ペプチド（ＭＰＰ）であり、Ｌは、ＡとＢとを結合させる共有結合またはリンカーペプチドのいずれかであり、Ｂは、カチオン性ポリペプチド、カチオン性部分、またはカチオン性部分に共有結合したカチオン性ペプチドのいずれかであり、ここで、非天然のペプチドは、トランスフェクション複合体の構成成分としての非天然のペプチドの存在が、トランスフェクション複合体のトランスフェクション効率を、非天然のペプチドを含まない同一のトランスフェクション複合体のものよりも最大１０％、最大１５％、２０％、最大２５％、最大３０％、最大３５％、最大４０％、最大４５％、最大５０％、最大５５％、最大６０％、最大６５％、最大７０％、最大７５％、最大８０％、最大８５％、最大９０％、最大９５％、最大１００％、最大１５０％、最大２００％、最大２５０％、最大３００％、最大３５０％、最大４００％、最大５００％、または５００％を上回って強化または改善するように増加させることを特徴とする。 In some embodiments, the transfection complex may comprise a lipid aggregate composition, wherein the lipid aggregate composition comprises at least one cationic lipid (possibly more than one cationic lipid). Including, optionally in contact with a cargo molecule in the presence of at least one helper lipid, the at least one non-natural peptide has the following general structure:

A is a membrane permeable peptide (MPP), L is either a covalent bond or a linker peptide that binds A and B, and B is a cationic polypeptide, cationic moiety, or cationic moiety A non-natural peptide, wherein the presence of the non-natural peptide as a component of the transfection complex reduces the transfection efficiency of the transfection complex. Up to 10%, up to 15%, 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50% than those of the same transfection complex without the natural peptide Up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, Increased to strengthen or improve over 100%, up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 500%, or 500% To do.

  A can be any peptide, which is not limited and is independent of the mechanism by which the peptide performs its function, eg a cargo molecule as defined above, in particular DNA or RNA. A nucleic acid molecule such as, for example, from a cell culture medium or an extracellular compartment such as intestinal fluid or body fluid, the cargo molecule is carried into the cytoplasmic compartment of the cell where it achieves at least one measurable biological response or function It has been known or demonstrated to enhance or facilitate transport across cell membranes. The determination of what constitutes an “enhancement” of transport across the cell membrane is well within the skill of one of ordinary skill in the art, and suitable peptides or known ones that function to enhance or facilitate the transport of cargo molecules in such a manner The identification of peptide variants of this is readily apparent to those skilled in the art using a wide range of known techniques.

  In some non-limiting embodiments, the peptide sequence of A has about 5 to 75 amino acids, about 5 to about 60 amino acids, about 5 to about 50 amino acids, about 5 to about 40 amino acids. Amino acids, about 5 to about 30 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, about 10 to about 75 amino acids, about 10 to about 60 amino acids, about 10 Can be about 50 amino acids, about 10 to about 40 amino acids, about 10 to about 30 amino acids, about 10 to about 20 amino acids, or about 10 to about 15 amino acids, wherein A The presence of the non-natural peptide as a component of the transfection complex makes the transfection efficiency of the transfection complex up to 10% higher than that of the same transfection complex without the non-natural peptide. Max 15%, 20%, Max 25%, Max 30%, Max 35%, Max 40%, Max 45%, Max 50%, Max 55%, Max 60%, Max 65%, Max 70%, Max 75% Up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, up to 150%, up to 200%, up to 250%, up to 300%, up to 350%, up to 400%, up to 500%, or It is characterized by strengthening over 500%.

  Various peptide sequences suitable for use as MPPs in the non-natural peptides described herein (ie region A of structure A-L-B or B-L-A shown above) Any of those known in the art can be used in the practice of the present invention without limitation. A representative but non-limiting set of peptides known to function as MPPs is shown in Table 1.

  In some non-limiting embodiments, L is a peptide comprising a peptide sequence selected from any one of SEQ ID NOs: 1-68, or at least 50% in any one of SEQ ID NOs: 1-68, Cargo into the interior of a cell having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence similarity At least 50% of its ability to enhance delivery of the molecule at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 155%, over 100%, up to 115%, up to 120%, up to 130%, Large 140%, up to 150%, up to 160% up to 170%, to retain the 180% maximum, its variants.

  In some embodiments, L can be a covalent bond that joins A and B.

  In some embodiments, L may comprise a neutral (uncharged at physiological pH) dipeptide, wherein any one of the two amino acids in the dipeptide comprises at least one polar side chain. . In certain embodiments, L may comprise a dipeptide comprising at least one polar side chain or at least one hydrophobic side chain, wherein the polar or hydrophobic side chain is preferably a bulky side chain. Absent. In certain embodiments, L may comprise a dipeptide comprising at least one glycine, at least one valine, at least one alanine, at least one serine, or at least one threonine. In some embodiments, L may comprise a dipeptide selected from the list consisting of GG, AA, GA, AG, AS, AY, GS, GT, GV, AV, SV, TV, VG, VA, and VT. Good.

  In some embodiments, L is from about 3 to about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 15, about 14, about 13, about 12, about 11, about May be a linker peptide having 10, about 9, about 8, about 7, about 6, about 5, about 4 amino acids, wherein at least about 50%, at least about 60%, at least about More than 70%, at least about 80%, at least about 90%, or greater than about 90% are neutral.

  In some embodiments, L is about 3 to about 50, about 5 to about 25, about 6 to about 20, about 8 to about 15 amino acids, or about 4, 5, 6, 7, 8, 9 Linker peptide having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids Wherein up to about 35% of the amino acids contain neutral polar side chains and / or at least 35% of the amino acids contain hydrophobic side chains, polar and hydrophobic The side chain is not a bulky side chain.

  In some embodiments, L is about 3 to about 50, about 5 to about 25, about 6 to about 20, about 8 to about 15 amino acids, or about 4, 5, 6, 7, 8, 9 Linker peptide having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids Wherein up to about 35% of the amino acids are selected from serine, threonine, valine, isoleucine, and leucine.

  In some embodiments, L is about 3 to about 50, about 5 to about 25, about 6 to about 20, about 8 to about 15 amino acids, or about 4, 5, 6, 7, 8, 9 Linker peptide having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids Wherein at least about 50%, at least about 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or more of the amino acid is glycine or alanine It is.

式中、各Ｘは、独立して、非極性側鎖を有する中性アミノ酸であり、各Ｙは、独立して、極性側鎖を有する中性アミノ酸であり、ｍは３〜１０の整数であり、ｎは１〜５の整数であり、Ｌが結合でない場合、ｐは１〜２０の整数である。ある実施形態において、ｍ＞ｎである。一部の実施形態では、ｍが２でありｎが１であるか、またはｍが３でありｎが１もしくは２である。一部の実施形態では、各Ｘは、独立して、グリシン、アラニン、バリン、ロイシン、またはイソロイシンである。一部の実施形態では、各Ｙは、独立して、セリンまたはスレオニンである。 In some embodiments, L may be a linker peptide having the structure:

In the formula, each X is independently a neutral amino acid having a nonpolar side chain, each Y is independently a neutral amino acid having a polar side chain, and m is an integer of 3 to 10. Yes, n is an integer from 1 to 5, and when L is not a bond, p is an integer from 1 to 20. In some embodiments, m> n. In some embodiments, m is 2 and n is 1, or m is 3 and n is 1 or 2. In some embodiments, each X is independently glycine, alanine, valine, leucine, or isoleucine. In some embodiments, each Y is independently serine or threonine.

  Various peptide sequences suitable for use as a linker (ie, region L of structure A-LB or B-LA shown above) in the non-natural peptides described herein are: Any of these can be used in the practice of the invention, and any of them can be used in the practice of the invention without limitation. A representative but non-limiting set of linker peptides contemplated for use in the embodiments described herein is set forth in Table 2.

  In some non-limiting embodiments, L is a peptide comprising a peptide sequence selected from any one of SEQ ID NOs: 69-81, or at least 50% in any one of SEQ ID NOs: 69-81, Cargo into the interior of a cell having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence similarity At least 50% of its ability to enhance delivery of the molecule at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 155%, over 100%, up to 115%, up to 120%, up to 130 , Up to 140% up to 150%, up to 160% up to 170%, to retain the 180% maximum, its variants.

  In some embodiments, B can be either a cationic polypeptide, a cationic moiety, or a cationic peptide covalently linked to the cationic moiety. Any cationic moiety known in the art to impart a cationic charge to the molecule, particularly peptides, can be selected for use in the present invention without limitation. Preferred but non-limiting examples of cationic moieties suitable for use in the present invention include polyamines such as one or more of putrescine, cadaverine, spermine, spermidine. Additional cationic moieties can include poly-L-lysine.

  In some embodiments, B is about 3 to about 50 amino acids, about 5 to about 25, about 6 to about 20, about 8 to about 15 amino acids, or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids It may be a peptide having a peptide sequence, wherein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% of this amino acid Or at least 95% are positively charged at physiological pH.

Various peptide sequences suitable for use as the cationic region (ie, region B of structure A-L-B or B-LA shown above) in the non-natural peptides described herein are: Can be used in the practice of the present invention, any of which can be used in the practice of the present invention without limitation. A representative but non-limiting set of linker peptides contemplated for use in the embodiments described herein is set forth in Table 3.

Table 4 lists various peptide sequences that can be used in the practice of the present invention, but the list of peptide sequences in Table 4 is merely exemplary, and the scope of the present invention is limited only to those explicitly stated. One skilled in the art will understand that this is not meant to be limiting. Conversely, the possibility of a large number of peptides potentially useful in the practice of the invention described herein is possible based on the above teachings regarding regions A, L, and B of the peptides of the invention. It will be readily apparent to the merchant. Furthermore, it is well within the skill of one of ordinary skill in the art to determine whether a given peptide is within the scope of the present invention using standard techniques in the art without undue experimentation. Is within the range. In addition, it will be understood that various variants of the peptide sequences listed in Table 4 are also within the scope of the present invention so long as such variants meet the structural and functional characteristics described above. . Variants of the peptide sequences listed in Table 4 or any other candidate peptide variants not explicitly listed in Table 4 but fulfilling the structural and functional requirements described above include deletions, insertions, and Substitution with natural or non-proteinogenic amino acids may be included.

  In some embodiments, the peptide sequence of A is from 5 to about 50 amino acids, and A provides at least 10% or more delivery of the molecule into the cell in the presence of a cationic lipid-based transfection reagent. At least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% At least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 200%, at least 250%, At least 300%, at least 350% At least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, at least 1500%, or at least 2000% And

  In some embodiments, A is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least at any one of the peptide sequences listed in Table 1. 80%, at least 85%, at least 90%, at least 95% similar and A is at least 10% or more, at least 15% or more, at least 20% or more, at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more, at least 90% or more, low At least 95%, at least 100%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, At least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

  In some embodiments, A is any one or more of the peptide sequences set forth in SEQ ID NOs: 1-68, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar and at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or more of its activity At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , At least 85% or more, at least 90% Above, at least 95%, at least 100%, at least 200% or more, at least 250% or more, at least 300% or more, at least 350% or more, at least 400% or more, at least 500% or more, at least 600% or more, at least 700% or more Variants having at least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

  In some embodiments, A is any one or more of the peptide sequences set forth in SEQ ID NOs: 14-35, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar and at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or more of its activity At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , At least 85% or more, at least 90 Or more, at least 95% or more, at least 100%, at least 200% or more, at least 250% or more, at least 300% or more, at least 350% or more, at least 400% or more, at least 500% or more, at least 600% or more, at least 700% or more Variants having at least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

  In some embodiments, A is any one or more of the peptide sequences set forth in SEQ ID NOs: 37-43, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar and at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or more of its activity At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , At least 85% or more, at least 90 Or more, at least 95% or more, at least 100%, at least 200% or more, at least 250% or more, at least 300% or more, at least 350% or more, at least 400% or more, at least 500% or more, at least 600% or more, at least 700% or more Variants having at least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

  In some embodiments, A is any one or more of the peptide sequences set forth in SEQ ID NOs: 44, 45, 46, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, At least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar, at least 10% or more of the activity, at least 15% or more, at least 20% or more, at least 25% or more, at least 30 %, At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% % Or more, at least 85% or more, at least 90% or more, at least 95% or more, at least 100%, at least 200% or more, at least 250% or more, at least 300% or more, at least 350% or more, at least 400% or more, at least 500% or more, at least 600% or more, at least 700 % Or more, at least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

  In some embodiments, A is any one or more of the peptide sequences set forth in SEQ ID NOs: 52-68, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar and at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or more of its activity At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , At least 85% or more, at least 90 Or more, at least 95% or more, at least 100%, at least 200% or more, at least 250% or more, at least 300% or more, at least 350% or more, at least 400% or more, at least 500% or more, at least 600% or more, at least 700% or more Variants having at least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

  In some embodiments, A is any one or more of the peptide sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID NO: 14, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar and at least 10% or more of its activity, At least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, At least 65%, at least 70%, at least 75% At least 80% or more, at least 85% or more, at least 90% or more, at least 95% or more, at least 100%, at least 200% or more, at least 250% or more, at least 300% or more, at least 350% or more, at least 400% or more, at least It may comprise variants thereof having 500% or more, at least 600% or more, at least 700% or more, at least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

  In one embodiment of the invention, the non-natural peptide A is any one or more of the peptide sequences listed in Table 4, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70% , At least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar and at least 10%, at least 15%, at least 20%, at least 25%, at least 30% of the activity At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% Or more, at least 85% or more, at least 90% or more, at least 95% or more, at least 100%, at least 200% or more, at least 250% or more, at least 300% or more, at least 350% or more, at least 400% or more, at least 500% or more, at least 600% or more, at least 700 % Or more, at least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

  In one aspect of the invention, the non-natural peptide A is any one or more of the peptide sequences set forth in SEQ ID NOs: 89-107, or at least 50%, at least 55%, at least 60%, at least 65%, At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar, at least 10% or more of its activity, at least 15% or more, at least 20% or more, at least 25% or more, At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85 Or more, at least 90% or more, at least 95% or more, at least 100%, at least 200% or more, at least 250% or more, at least 300% or more, at least 350% or more, at least 400% or more, at least 500% or more, at least 600% or more , Having at least 700% or more, at least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

  In one aspect of the invention, the non-natural peptide A is any one or more of the peptide sequences set forth in SEQ ID NOs: 89-96, or at least 50%, at least 55%, at least 60%, at least 65%, At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar, at least 10% or more of its activity, at least 15% or more, at least 20% or more, at least 25% or more, At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85% Above, at least 90%, at least 95%, at least 100%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600% , Having at least 700% or more, at least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

  In one aspect of the invention, the non-natural peptide A is any one or more of the peptide sequences set forth in SEQ ID NOs: 89, 92, 98, 103, or 106, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% similar, at least 10% or more, at least 15% or more, at least 20% of its activity At least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% Or more, at least 75% or more, at least 80% or more, at least 85% or more, at least 90% or more, at least 95% or more, at least 100%, at least 200% or more, at least 250% or more, at least 300% or more, at least 350% or more, at least 400% or more, at least 500 % Or more, at least 600% or more, at least 700% or more, at least 800% or more, at least 900% or more, at least 1000% or more, at least 1500% or more, or at least 2000% or more.

Transfection enhancers Complexes formed between unnatural peptides, nucleic acids, and transfection agents serve as nuclear or other intracellular translocations, serve as transport or transport, and are receptor ligands Proteins or peptides, including cell adhesion signals, cell targeting signals, cell internalization signals, or endocytosis signals, and viral fusion-inducible proteins, enveloped viruses, viral nuclear internalization signals, receptor ligands, cell adhesion signals, It can be further enhanced by including portions such as cellular targeting signals, or peptides or functional portions thereof that trigger internalization or endocytosis.

  The complex is also a nuclear internalization protein or peptide, a fusion-inducible peptide or protein, a receptor ligand peptide or protein, a transport peptide or protein, or a viral peptide that is distinctly different in amino acid sequence from the non-natural peptide of the invention Alternatively, transfection enhancing agents such as proteins can optionally be included. Suitable viral peptides can be derived from viruses such as influenza virus, vesicular stomatitis virus, adenovirus, alphavirus, Semliki Forest virus, hepatitis virus, HIV virus, or simian virus. Transfection enhancers also include, for example, insulin, transferrin, epidermal growth factor, fibroblast growth factor, cell-targeted antibody, lactoferrin, fibronectin, adenovirus penton base, knob, hexon protein, vesicular stomatitis virus glycoprotein, Semliki Forest virus core protein, influenza hemagglutinin, hepatitis B core protein, HIV Tat protein, herpes simplex virus VP22 protein, histone protein, arginine rich cell permeability protein, high mobility protein, and invesin protein, and internalin protein, Endotoxin, diphtheria toxin, Shigella toxin, melittin, magainin, gramicidin, cecrofin, defensin, prote Phosphorus, tachyplesin, thionine, indolicidin, bactenecin, drosomycin, apidaecin (apidaecin), cathelicidin, a protein that increases the permeability of the bacterial, nisin, buforin (buforin), or a fragments thereof. The transfection enhancing agent can be chloroquine, a lysosome-tropic compound, or any derivative, variant, or combination thereof. Transfection agents may contain multimers of the same or different peptides or proteins.

  Any protein or peptide (or fragment or portion thereof) of the present invention can be used according to the present invention alone or in combination with other proteins or peptides. In preferred embodiments, two or more, three or more, four or more, five or more, six or more proteins and / or peptides are used in the present invention. Furthermore, such single or multiple proteins and / or peptides are used in combination with one or more, two or more, three or more, four or more, five or more, six or more transfection agents, etc. May be. In another preferred embodiment, at least two peptides and / or proteins are used in combination with a transfection agent, preferably at least two transfection agents such as lipids and / or polycations such as dendrimers or PEI.

  A further embodiment of the present invention provides a transfectant comprising a non-natural peptide as described above, in combination with one or more transfection reagents that may comprise one or more cationic lipids, and optionally one or more helper lipids. The target complex. In some embodiments, the transfection complex may include cargo delivered to the interior of the cell, or may optionally be administered to an animal or human patient that would benefit from its administration. Preferred but non-limiting cargo molecules suitable for use in the present invention include nucleic acid molecules such as DNA molecules or RNA molecules. Suitable DNA molecules can include DNA molecules having an expressible nucleic acid sequence, such as an expression vector, or cDNA molecules comprising an open reading frame encoding a protein. Other suitable molecules that can function as suitable cargo molecules in the practice of the present invention include RNA molecules such as mRNA molecules or RNAi molecules.

Methods for making peptides:
The non-natural peptides of the present invention can be produced by known peptide synthesis methods known to those skilled in the art including, without limitation, recombinant methods or peptide synthesis chemistry, eg, solid phase peptide synthesis. It can be noted that the solid phase synthesis method (Marrifield, J. Am. Chem. Soc., 85, 2149-2154, 1963) is just one example of such a peptide synthesis method. At present, peptides can be simply generated in a relatively short period of time using an automated universal peptide synthesizer based on these principles. In addition, peptides can be generated using well-known recombinant protein techniques, which techniques are widely known to those skilled in the art.

Transfection Reagents The present invention is also non-covalent, non-natural peptides according to the invention as described above and incorporated herein, at least one cargo molecule as defined above and incorporated herein, and And a transfection complex comprising at least one transfection reagent defined herein and incorporated herein.

  In certain preferred but non-limiting embodiments, the transfection reagent selected for use in the practice of the present invention may include one or more cationic lipids. In some embodiments, the one or more cationic lipids can optionally include at least one, and optionally more than one, neutral or helper lipids.

  In some embodiments, the transfection reagent may include one or more lipids, which can be cationic lipids. In some embodiments, the transfection reagent may comprise a mixture of neutral and cationic lipids. In some embodiments, the transfection reagent may comprise one or more peptides and / or proteins, which are clearly special from the non-natural peptides of the present invention, and alone or one or more. Can be provided as a mixture with other lipids. As a non-limiting example, one such peptide can include, for example, reagents such as PLUS ™ reagent (Life Technologies, Carlsbad, Calif.). In some preferred embodiments, the transfection reagent is non-covalent with macromolecules / cargo delivered to the interior of the cell, non-natural peptides of the invention, and optionally one or more helpers or neutral lipids. Form a complex of bonds. In preferred embodiments, transfection complexes made according to the rough methods described herein have a net positive charge, thereby facilitating the interaction of the transfection complex with the cell membrane.

  In some embodiments, a transfection reagent suitable for use according to the present invention can be any material, formulation, or composition known to those of skill in the art that facilitates the entry of macromolecules into cells. In some embodiments, the transfection reagent can be any compound and / or composition that increases the uptake of one or more nucleic acids or other cargo molecules into one or more target cells.

Various transfection reagents are known to those skilled in the art. Suitable transfection reagents include cationic polymers such as polyethyleneimine (PEI), polymers of positively charged amino acids such as polylysine and polyarginine, positively charged and split dendrimers, cationic β-cyclodextrin One or more compounds and / or compositions, including but not limited to polymers (CD polymers), DEAE-dextran, and the like. In some embodiments, reagents for introduction of macromolecules into cells can include one or more lipids that can be cationic lipids and / or neutral lipids. Preferred lipids include N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylcholine (DOPE), 1,2-bis (ole). Oiloxy) -3- (4′-trimethylammonio) propane (DOTAP), 1,2-dioleoyl-3- (4′-trimethylammonio) butanoyl-sn-glycerol (DOTB), 1,2-dioleoyl- 3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl (4′-trimethylammonio) butanoate (ChoTB), cetyltrimethylammonium bromide (CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide ( DORI), 1,2-Geolay Oxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), O, O′-didodecyl-N- [p (2- Trimethylammonioethyloxy) benzoyl] -N, N, N-trimethylammonium chloride, spermine conjugated to one or more lipids (eg, 5-carboxyspermylglycine dioctadecylamide (DOGS), N, N ^I , N ^{II, N III} - tetramethyl ^{-N, N I, N II,} N III - tetra palmityl spermine (TM-TPS), and dipalmitoyl Rufasu phosphatidyl ethanolamine 5-carboxyfluorescein spelling mill amine de (DPPES)), lipoic Polylysine (DOP Polylysine), TRIS (tris (hydroxymethyl) aminomethane, tromethamine), and / or peptides such as trilysyl-alanyl-TRIS mono, di, and tripalmitate (3β- [N- -(N ', N'-dimethylaminoethane) -carbamoyl] cholesterol (DC-Chol), N- (α-trimethylammonioacetyl) -didodecyl-D-glutamate chloride (TMAG), dimethyldioctadecylammonium bromide (DDAB) ), 2,3-dioleyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), and combinations thereof, Not limited to

  One skilled in the art understands that certain combinations of the above lipids have been shown to be particularly suitable for introduction of nucleic acids into cells, eg, 3: 1 (by weight of DOSPA and DOPE / Weight) is a combination of Life Technologies Corporation, Carlsbad, Calif. Available under the trade name LIPOFECTAMINE ™ and a 1: 1 (weight / weight) combination of DOTMA and DOPE is available from Life Technologies Corporation, Carlsbad, Calif. Available under the trade name LIPOFECTIN® and a 1: 1 (M / M) combination of DMRIE and cholesterol is available from Life Technologies Corporation, Carlsbad, Calif. Available under the trade name DMRIE-C Reagent and a 1: 1.5 (M / M) combination of TM-TPS and DOPE is available from Life Technologies Corporation, Carlsbad, Calif. Available under the trade name CELLFECTIN® and a 1: 2.5 (weight / weight) combination of DDAB and DOPE is available from Life Technologies Corporation, Carlsbad, Calif. Is available under the trade name LIPFECTACE®. In addition to the lipid combinations described above, other formulations are known to those skilled in the art that include lipids in admixture with other compounds, particularly in admixtures with peptides and proteins containing nuclear localization sequences. . For example, see International Application No. PCT / US99 / 26825, published as International Publication No. WO 00/27795, both of which are incorporated herein by reference.

  Lipid aggregates such as liposomes have been found useful as drugs for delivery of macromolecules into cells. In particular, lipid aggregates comprising one or more cationic lipids have been shown to be very efficient in delivering anionic macromolecules (eg, nucleic acids) into cells. One widely used cationic lipid is N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA). Liposomes containing DOTMA alone or as a 1: 1 mixture with dioleoylphosphatidylethanolamine (DOPE) have been used to introduce acid into cells. A 1: 1 mixture of DOTMA: DOPE is available from Life Technologies Corporation, Carlsbad, Calif. Commercially available under the trade name LIPOFECTIN ™. Another cationic lipid that has been used to introduce nucleic acids into cells is 1,2-bis (oleoyl-oxy) -3--3- (trimethylammonia) propane (DOTAP). DOTAP differs from DOTMA in that the oleoyl moiety is bound to the propylamine backbone by an ether bond in DOTAP, while it is bound by an ester bond in DOTMA. DOTAP is seen to be more easily degraded by target cells. A structurally related group of compounds in which one of the methyl groups of the trimethylammonium moiety is replaced with a hydroxyl group is structurally similar to the Rosenthal inhibitor (RI) of phospholipase A (Rosenthal, et al., (1960) J. Biol. Chem. 233: 2202-2206.). The RI has a stearoyl ester attached to a propylamine core. The dioleoyl analog of RI is generally abbreviated as DOR1-ether and DOR1-ester, depending on the linkage between the lipid moiety and the propylamine core. The hydroxyl group of the hydroxyethyl moiety may be further derivatized with carboxyspermine, for example by esterification.

  Another class of compounds that have been used to introduce macromolecules into cells contains a carboxyspermine moiety bound to a lipid (Behr, et al., (1989) Proceedings of the National Academy of Sciences, USA 86. : 6982-6986 and European Patent 0 394 111). Examples of this type of compound include dipalmitoyl phosphatidylethanolamine 5-carboxyspermylamide (DPPES) and 5-carboxyspermylglycine dioctadecylamide (DOGS). DOGS is available from PROMEGA ™, Madison, Wis. Commercially available under the trade name TRANSFECTAM ™.

  Cationic derivatives of cholesterol (3β- [N- (N ′, N′-dimethylaminoethane) -carbamoyl] cholesterol, DC-Chol) have been synthesized and formulated into liposomes along with DOPE (Gao, et al (1991) BBRC 179 (1): 280-285.), Which has been used to introduce DNA into cells. It has been reported that liposomes formulated in this way efficiently introduce DNA into cells at low cytotoxic levels. Lipopolylysine (Zhou, et al., (1991) BBA 1065: 8-14) formed by conjugating polylysine to DOPE is effective in introducing nucleic acids into cells in the presence of serum. Has been reported.

  Other types of cationic lipids that have been used to introduce nucleic acids into cells include US Pat. Nos. 5,674,908 and 5,834,439, and International Publication No. WO 00/27795. Highly loaded polycationic ammonium, sulfonium, and phosphonium lipids such as those described in published International Application No. PCT / US99 / 26825 may be mentioned.

  One non-limiting transfection reagent for delivery of macromolecules according to the present invention is LIPOFECTAMINE 2000 ™ or a derivative thereof, which is available from Life technologies (International Publication No. WO 00/27795). (See US International Application No. PCT / US99 / 26825).

  Another preferred but non-limiting transfection reagent suitable for delivery of macromolecules to cells is EXPIFECTAMINE ™ or a derivative thereof.

  Other suitable transfection reagents include LIOFECTAMINE (R) RNAiMAX, LIPOFECTAMINE (R) LTX, OLIGOFECTAMINE (R), CELLFECTTIN (R), INVIVOFECTAMINE (R), INVIVOFECTAMINE (R), And any of the lipid reagents or formulations disclosed in U.S. Patent Application Publication No. 2012/0136073 (incorporated herein by reference) by Yang et al. A variety of other transfection reagents are known to those of skill in the art and can be evaluated for their suitability for the transient transfection systems and methods described herein.

  Various preferred but non-limiting cationic lipids and transfection reagents suitable for use in the present invention will now be described in more detail below. However, the explicit disclosure of one or more specific cationic lipids, or one or more genera of cationic lipids, does not address the use of other reagents or lipids that can be used with the non-natural peptides of the present invention. It is not meant to be excluded, and the selection of alternative cationic lipids or transfection reagents and their use in the context of the present invention are well within the skill of one skilled in the art, and It should be noted that those skilled in the art can readily use such reagents without departing from the spirit and scope of the present invention.

  Some embodiments of the present invention provide lipid aggregates comprising one or more non-natural peptides described above in combination with one or more cationic lipids. It is not limited or constrained to any theory or mechanical explanation regarding the ability of the composition to form the basis of the present invention, and is intended only to provide a complete disclosure thereof. A non-natural peptide is a transfection complex comprising a lipid aggregate and a cargo molecule that is delivered to the interior of a cell when used in combination with one or more transfection reagents, particularly one or more cationic transfection lipids. It is thought to improve the ability. The use of cationic lipids, optionally with one or more helper lipids or one or more neutral lipids, may allow better encapsulation of cargo molecules by lipid aggregates, and also liposomal lipid aggregates Can help fusion with the target cell membrane, thereby enhancing the delivery of cargo molecules.

  Cationic lipids useful for use in forming the transfection complexes of the present invention can be either monovalent or multivalent cationic lipids or mixtures of cationic lipids. Recognized in the art as useful in transfection methods, including but not limited to DOTMA, DOTAP, DDAB, DMRIE, DOSPA, DOSPER, TMTPS, DHMS, DHDMS, and analogs or homologues thereof Of particular interest are cationic lipids. In some cases, the lipid aggregates may further comprise at least one additional helper lipid. Helper lipids are known in the art and preferably include, but are not limited to, neutral lipids selected from the group consisting of DOPE, DOPC, and cholesterol. In some cases, the transfection complexes of the present invention may comprise Lipofectin®, LIPOFECTAMINE ™ RNAiMAX and LIPOFECTAMINE ™ 2000, LIPOFECTAMINE® 3000, LIPOFECTAMINE® LTX (Life TecorCorfeCorminCortex). , CA.), or commercially available transfection reagents containing cationic lipids.

  Further embodiments of the invention include one or more cationic lipids, optionally one or more helper lipids, and one or more complexed with one or more of the non-natural peptides described above. Cationic lipid aggregates comprising cargo molecules are provided. The cargo molecule can be any substance that is transported inside the cell, either in culture in the laboratory or in tissue in animals or humans. The cargo may be a macromolecule such as a nucleic acid, protein, or peptide, or a drug or small organic molecule, depending on the application. In some embodiments, preferred cargoes for forming the transfection complex are nucleic acids such as, for example, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In some embodiments, the preferred cargo may be a DNA molecule. The DNA can be either linear DNA or circular DNA, for example, DNA in the form of a circular plasmid, episome, or expression vector. In certain preferred but non-limiting embodiments, the term macromolecule comprises at least one operably linked to one or more nucleic acid sequences required for transcription of mRNA from an expressible nucleic acid sequence. Refers to complementary DNA (cDNA) having an expressible nucleic acid sequence, including an open reading frame. In other embodiments, the preferred cargo may be an RNA molecule. RNA molecules are known to those skilled in the art without limitation including, but not limited to, mRNA, siRNA, miRNA, antisense RNA, ribozyme, or any other type or species of RNA molecule. Any type of RNA molecule that is required to be delivered to the interior of the cell.

  Preferably, the transfection complex of the present invention may comprise a non-natural peptide as described above, at least one cargo, at least one cationic lipid, and optionally at least one helper lipid. Once formed, the transfection complex is stable in aqueous solution and is contacted with human or animal cells or tissues immediately after formation or stored for a period of time prior to contact with cells or tissues. Can do either. The transfection complex is stable and is at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, At least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 It can be stored for a period of at least 6 months, or at least 1 year. It is understood that the storage period may be any of these periods, for example, 31 minutes to 1 hour, or 1 hour to 24 hours.

  In general, the transfection complexes of the present invention can comprise any cationic lipid, either monovalent or multivalent, including those contained in known transfection reagents (see Table 5). Cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof. Linear and branched alkyl and alkene groups of cationic lipids can contain from 1 to about 25 carbon atoms. Preferred linear or branched alkyl or alkene groups have 6 or more carbon atoms. More preferred linear or branched alkyl or alkene groups have from 8 to about 20 carbon atoms. The alicyclic group may contain about 6-30 carbon atoms, more preferably 8-20 carbon atoms. Preferred alicyclic groups include cholesterol and other steroid groups. Cationic lipids are prepared with various counterions (anions) including, among others, Cl-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, triflate, and nitrate. Can be done.

  In the lipid aggregates of the present invention, cationic lipids are combined with non-cationic lipids, preferably neutral lipids, to form lipid aggregates that bind to the modified peptide-nucleic acid complex. Neutral lipids useful as helper lipids in the present invention include, inter alia, lecithin (and derivatives thereof); phosphotidylethanolamine (and derivatives thereof); phosphatidylethanolamine, such as DOPE (dioleoylphosphatidylethanolamine). , DphPE (diphytanoylphosphatidyl-ethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), dipalmiteoylphosphatidyl-ethanolamine, POPE (palmitoyloleoylphosphatidylethanolamine), and distearoylphosphatidylylamine Ethanolamine; phosphotidylcholine; phosphatidylcholine such as DOPC (di- Oleoylphosphidylcholine), DPPC (dipalmitoylphosphatidylcholine), POPC (palmitoyloleoylphosphatidylcholine), and distearoylphosphatidylcholine; phosphatidyl-glycerol; phosphatidylglycerol, eg, DOPG (dioleoylphosphatidylglycerol), PG Dipalmitoylphosphatidylglycerol), and distearoylphosphatidylglycerol; phosphatidyl-serine (and derivatives thereof); phosphatidylserine, such as dioleoyl- or dipalmitoylphosphatidylserine; diphosphatidylglycerol; fatty acid ester; glycerol ester; sphingolipid; Pin (cardolipin); cerebrosides; and ceramides; and mixtures thereof. Neutral lipids also include cholesterol and other 3βOH-sterols, and derivatives thereof.

The following patent documents, patent applications, or references are described in their entirety, and in particular, neutral (helper) lipids that can be used to construct the lipid aggregates of the present invention with cationic lipids and cationic lipids. No. 6,075,012, US Pat. No. 6,020,202, US Pat. No. 5,578,475, US Pat. No. 5,578,475, the disclosures of which are incorporated herein by reference. , 736,392, 6,051,429, 6,376,248, 5,334,761, 5,316,948, 5,674,908 No. 5,834,439, No. 6,110,916, No. 6,399,663, No. 6,716,882, No. 5,627,159, International Publication No. WO04063342 A2 PCT / US / 2004/000430 published as PCT / US / 9926825 published as International Publication No. WO0027795 A1, PCT / US / 04016406 published as International Publication No. WO04105697, And PCT / US2006 / 019356 published as International Publication No. WO07130073 A2. Table 5 also lists transfection agents comprising neutral lipids that can be used to construct the lipid aggregates of the present invention with cationic lipids and cationic lipids.

及びその塩を有し、式中、
Ｒ_１及びＲ_２は、独立して、８〜３０個の炭素原子を有するアルキル、アルケニル、もしくはアルキニル基、
８〜３０個の炭素原子を有し、アルコール、アミンアルコール、アミン、アミド、エーテル、ポリエーテル、エステル、メルカプタン、アルキルチオ、もしくはカルバモイル基のうちの１つ以上で任意に置換されたアルキル、アルケニル、もしくはアルキニル基であるか、またはＲ_１が−（ＣＨ_２）_ｑ−Ｎ（Ｒ_６）_ｔＲ_７Ｒ_８であり、
Ｒ_３及びＲ_４は、独立して、水素、または８〜３０個の炭素原子を有し、アルコール、アミノアルコール、アミン、アミド、エーテル、ポリエーテル、エステル、メルカプタン、アルキルチオ、もしくはカルバモイル基のうちの１つ以上で任意に置換された、アルキル、アルケニル、もしくはアルキニル基であり、
Ｒ_５〜Ｒ_８は、独立して、水素、またはアルキル、アルケニル、もしくはアルキニル基であり、
Ｒ_９は、水素、またはアルキル、アルケニル、もしくはアルキニル基、炭水化物、またはペプチドであり、
ｒ、ｓ、及びｔは、１または０であり、示されるＲ基の存在または不在を示し、ｒ、ｓ、またはｔのうちのいずれかが１である場合、示されるＲ基が結合する窒素は、正に荷電しており、ｒ、ｓ、またはｔのうちの少なくとも１つが１であり、
ｑは、１以上６以下の範囲の整数であり、
Ｘ^ｖ−は、アニオンであり、Ｖはアニオンの価数であり、Ａはアニオンの数であり、
Ｌは、２つの窒素を共有結合させることができる、（ＣＨ_２）_ｎから選択される二価の有機ラジカルであり、ここで、ｎは、１以上１０以下の範囲の整数であり、これは、１つ以上のＺＲ_１０基で任意に置換され、ＺはＯまたはＳであり、Ｒ_１０は、水素、またはアルキル、アルケニル、もしくはアルキニル基であるか、あるいは
｛−（ＣＨ_２）_ｋ−Ｙ−（ＣＨ_２）_ｍ｝_ｐ−であり、ｋ及びｍは、独立して、１以上１０以下の範囲の整数であり、ｐは１以上６以下の範囲の整数であり、Ｙは、Ｏ、Ｓ、ＣＯ、ＣＯＯ、ＣＯＮＲ_１１、ＮＲ_１１ＣＯ、またはＮＲ_１１ＣＯＲ_１１Ｎであり、Ｒ_１１は、いずれの他のＲ_１１からも独立して、水素またはアルキル基であり、
Ｒ_１〜Ｒ_１０のアルキル、アルケニル、またはアルキニル基の１つ以上のＣＨ_２基は、Ｏ、Ｓ、Ｓ−Ｓ、ＣＯ、ＣＯＯ、ＮＲ_１２ＣＯ、ＮＲ_１２ＣＯＯ、またはＮＲ_１２ＣＯＮＲ_１２で置き換えられてもよく、Ｒ_１２は、いずれの他のＲ_１２からも独立して、水素、またはアルキル、アルケニル、もしくはアルキニル基であり、
Ｒ_１〜Ｒ_１２のアルキル、アルケニル、またはアルキニル基は、１つ以上のＯＲ_１３、ＣＮ、ハロゲン、Ｎ（Ｒ_１３）_２、ペプチド、または炭水化物基で任意に置換され、Ｒ_１３は、他のＲ_１３から独立して、水素、またはアルキル、アルケニル、もしくはアルキニル基であり、
Ｒ_３及びＲ_４のうちの少なくとも１つは、アルキル基として存在する場合、ＯＲ_１３及びＮ（Ｒ_１３）_２基のいずれでも置換される。 In some preferred but non-limiting embodiments, the lipid aggregate may comprise at least a first cationic lipid and optionally at least a first neutral lipid, wherein the lipid aggregate The aggregate is suitable for forming a cationic complex with a nucleic acid under aqueous solution conditions, and the cationic lipid has the following structure:

And a salt thereof, wherein
R ₁ and R ₂ are independently alkyl, alkenyl or alkynyl groups having 8 to 30 carbon atoms,
Alkyl, alkenyl, having 8 to 30 carbon atoms, optionally substituted with one or more of alcohol, amine alcohol, amine, amide, ether, polyether, ester, mercaptan, alkylthio, or carbamoyl group, Or an alkynyl group, or R ₁ is — (CH ₂ ) _q —N (R ₆ ) _t R ₇ R ₈ ,
R ₃ and R ₄ are independently hydrogen or 8 to 30 carbon atoms, among alcohol, amino alcohol, amine, amide, ether, polyether, ester, mercaptan, alkylthio, or carbamoyl group An alkyl, alkenyl, or alkynyl group, optionally substituted with one or more of
R _{5 to} R ₈ are independently hydrogen or an alkyl, alkenyl, or alkynyl group,
R ₉ is hydrogen or an alkyl, alkenyl, or alkynyl group, carbohydrate, or peptide;
r, s, and t are 1 or 0, indicating the presence or absence of the indicated R group, and when either r, s, or t is 1, the nitrogen to which the indicated R group is attached Is positively charged and at least one of r, s, or t is 1,
q is an integer in the range of 1 to 6,
X ^v− is an anion, V is the valence of the anion, A is the number of the anion,
L is a divalent organic radical selected from (CH ₂ ) _n capable of covalently bonding two nitrogens, where n is an integer ranging from 1 to 10, Optionally substituted with one or more ZR ₁₀ groups, Z is O or S, R ₁₀ is hydrogen or an alkyl, alkenyl, or alkynyl group, or {— (CH ₂ ) _k —Y _{- (CH 2) m} p} - a and, k and m are independently an integer ranging from 1 to 10, p is an integer ranging from 1 to 6, Y is, O, is _{S, CO, COO, CONR 11} , NR 11 CO or _{NR 11 COR 11 N,, R} 11 , independently from any other _{R 11,} is hydrogen or an alkyl group,
Alkyl of R ₁ to R _10, alkenyl or one or more CH ₂ groups of alkynyl groups, replaces _{O, S, S-S,} CO, COO, NR 12 CO, NR 12 COO , or _NR 12 CONR _12, R ₁₂ , independently of any other R ₁₂ , is hydrogen or an alkyl, alkenyl, or alkynyl group;
The alkyl, alkenyl, or alkynyl group of R ₁ -R ₁₂ is optionally substituted with one or more OR ₁₃ , CN, halogen, N (R ₁₃ ) ₂ , peptide, or carbohydrate groups, wherein R ₁₃ is other Independently of R _{13 is} hydrogen or an alkyl, alkenyl, or alkynyl group;
At least one of R ₃ and R ₄ , when present as an alkyl group, is substituted with either an OR _{13 or} N (R ₁₃ ) ₂ group.

  The synthesis of these compounds and methods for preparing lipid aggregates incorporating them can be accomplished by any means known to those of skill in the art without limitation. Exemplary but non-limiting methods for synthesizing such compounds and methods for forming lipid aggregates incorporating them are described, for example, in US Pat. No. 7,166,745 and PCT Publication No. Which can be found in WO 00/27795, all of which are expressly incorporated by reference in their entirety as if fully set forth herein.

  In some embodiments, the lipid aggregate forming the basis of the present invention is further one selected from the list consisting of TMTPS, DOGS, DPPES, DOTMA, DOTAP, DDAB, DMRIE, DOSPA, and DOSPER, Some may optionally include more than one additional cationic lipid.

In some embodiments of the present lipid aggregates, particularly preferred but non-limiting cationic lipids used to form the transfection complexes of the present invention are dihydroxyl-dimyristyl spermine tetrahydrochloride having the following structure: (Hereinafter referred to as “DHDMS”).

In some embodiments of the present lipid aggregates, particularly preferred but non-limiting cationic lipids used to form the transfection complexes of the present invention are hydroxyl-dimyristyl spermine tetrahydrochloride ( Hereinafter referred to as “HDMS”).

  In some embodiments, the neutral lipid can be selected from: DOPE, cholesterol, or DOPC. In one embodiment, the neutral lipid can be one of cholesterol, DOPE, or DOPC. In certain embodiments, the lipid is cholesterol. In certain embodiments, the neutral lipid is DOPE. In certain embodiments, the lipid is DOPC.

  In certain embodiments, the optional second neutral lipid can be one of cholesterol, DOPE, or DOPC, but the second neutral lipid and the first neutral lipid are not the same. except for. In certain embodiments, the optional second neutral lipid is cholesterol. In certain embodiments, the optional second neutral lipid is DOPE. In certain embodiments, the optional second neutral lipid is DOPC.

  In some embodiments, the molar ratio of the cationic lipid in the lipid aggregate can range from about 0.1 to about 0.8. In some embodiments, the molar ratio of the cationic lipid in the lipid aggregate is from 0.1 to about 0.2, from about 0.15 to about 0.25, from about 0.2 to about 0.3, about 0.25 to about 0.35, about 0.3 to about 0.4, about 0.35 to about 0.45, about 0.4 to about 0.5, about 0.45 to about 0.55, about 0.5 to about 0.6, about 0.55 to about 0.65, about 0.6 to about 0.7, about 0.65 to about 0.75, about 0.7 to about 0.8, or It can be about 0.75 to about 0.85.

  In some embodiments, the molar ratio of DHDMS in the lipid aggregate can range from about 0.1 to about 0.7. In some embodiments, the molar ratio of cationic lipids in the lipid aggregate is about 0.1, about 0.2, about 0.25, about 0.3, or about 0.4, or between Can be any range included.

  In some embodiments, the molar ratio of DHDMS is from about 0.1 to about 0.4. In some embodiments, the molar ratio of DHDMS is about 0.1, about 0.2, about 0.25, about 0.3, or about 0.4, or any range included therebetween. is there.

  In some embodiments, the molar ratio of HDMS in the lipid aggregate can range from about 0.1 to about 0.4. In some embodiments, the molar ratio of the second cationic lipid in the lipid aggregate is about 0.1, about 0.2, about 0.25, about 0.3, or about 0.4, or It can be any range included between them.

  In some embodiments, the molar ratio of HDMS is from about 0.1 to about 0.4. In some embodiments, the HDMS molar ratio is about 0.1, about 0.2, about 0.25, about 0.3, or about 0.4, or any range included therebetween. is there.

  In some embodiments, the molar ratio of neutral lipids in the lipid aggregate can range from about 0.1 to about 0.8. In some embodiments, the molar ratio of the cationic lipid in the lipid aggregate is from 0.1 to about 0.2, from about 0.15 to about 0.25, from about 0.2 to about 0.3, about 0.25 to about 0.35, about 0.3 to about 0.4, about 0.35 to about 0.45, about 0.4 to about 0.5, about 0.45 to about 0.55, about 0.5 to about 0.6, about 0.55 to about 0.65, about 0.6 to about 0.7, about 0.65 to about 0.75, about 0.7 to about 0.8, or It can be about 0.75 to about 0.85, or any range therebetween.

  In some embodiments, the molar ratio of cholesterol is from about 0.1 to about 0.8. In some embodiments, the molar ratio of the cationic lipid in the lipid aggregate is from 0.1 to about 0.2, from about 0.15 to about 0.25, from about 0.2 to about 0.3, about 0.25 to about 0.35, about 0.3 to about 0.4, about 0.35 to about 0.45, about 0.4 to about 0.5, about 0.45 to about 0.55, about 0.5 to about 0.6, about 0.55 to about 0.65, about 0.6 to about 0.7, about 0.65 to about 0.75, about 0.7 to about 0.8, or It can be about 0.75 to about 0.85, or any range therebetween.

  In some embodiments, the molar ratio of DOPE is from about 0.1 to about 0.8. In some embodiments, the molar ratio of the cationic lipid in the lipid aggregate is from 0.1 to about 0.2, from about 0.15 to about 0.25, from about 0.2 to about 0.3, about 0.25 to about 0.35, about 0.3 to about 0.4, about 0.35 to about 0.45, about 0.4 to about 0.5, about 0.45 to about 0.55, about 0.5 to about 0.6, about 0.55 to about 0.65, about 0.6 to about 0.7, about 0.65 to about 0.75, about 0.7 to about 0.8, or It can be about 0.75 to about 0.85, or any range therebetween.

  In some embodiments, the molar ratio of DOPC is from about 0.1 to about 0.4. In some embodiments, the molar ratio of DOPC is about 0.1, about 0.2, about 0.25, about 0.3, or about 0.4, or any range included therebetween. is there.

  In some embodiments, the molar ratio of cholesterol is from about 0.2 to about 0.8. In some embodiments, the molar ratio of cholesterol is about 0.1, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0. .5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, or about 0.8, or any range included therebetween.

  In some embodiments, the molar ratio of DOPE is from about 0.2 to about 0.8. In some embodiments, the DOPE molar ratio is about 0.1, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0. .5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, or about 0.8, or any range included therebetween.

  In some embodiments, the molar ratio of DOPC is from about 0.2 to about 0.8. In some embodiments, the molar ratio of DOPC is about 0.1, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0. .5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, or about 0.8, or any range included therebetween.

  In some embodiments, the molar ratio of DHDMS is about 0.1, 0.2, 0.25, 0.3, 0.4, or 0.5 and the molar ratio of neutral lipids is about 0.1, 0.2, 0.25, 0.3, 0.4, or 0.5.

  In some embodiments, the HDMS molar ratio is about 0.1, 0.2, 0.25, 0.3, 0.4, or 0.5 and the neutral lipid molar ratio is about 0.1, 0.2, 0.25, 0.3, 0.4, or 0.5.

  Compositions of various lipid formulations according to some non-limiting embodiments of the present invention are provided in Table I. The provision of these exemplary embodiments is in no way meant to limit the scope of the invention to only the disclosed formulations. Conversely, it simply means providing a variety of potential lipid aggregate formulations that can be used in the practice of the present invention. In any case, these formulations may be altered or modified, additional components (eg, additional cationic or neutral lipids, peptide targeting moieties, etc.) may be added, or listed in Table I One of ordinary skill in the art will be able to optionally remove one of the listed neutral lipids and that the resulting formulation is within the spirit and scope of the invention described herein. Will be clear.

Preparation and Use of Complexes Containing Non-Natural Peptides Another embodiment of the present invention provides a method for delivering a polyanion, such as a nucleic acid molecule, to a cell (s), the method comprising one or more The lipid aggregate comprising a cationic lipid and one or more neutral lipids, preferably forming a liposome, and the cationic region B present therein, makes the lipid aggregate non-natural peptide of the present invention. In contact with the polyanion already complexed, thereby forming a neutral or positively charged polyanion-peptide-lipid aggregate complex, and incubating the cell (s) with this complex And including. Useful anions include proteins, peptides, and nucleic acids, preferably DNA or RNA. Preferably, the lipid aggregate further comprises at least one helper lipid. In some cases, the polyanion-lipid aggregate complex is stored for a period of time before being contacted with the cell (s). The polyanion-lipid aggregate complex is stable and is at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, At least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 It can be stored for months, at least 6 months, or at least 1 year, or any of these periods. The present invention is particularly useful for delivering RNAi, including siRNA, short hairpin RNA (shRNA), and small transiently regulated RNA (stRNA), which are optionally chemically modified.

  The method of the invention involves contacting any cell, preferably a eukaryotic cell, with a transfection complex comprising at least one non-natural peptide, a transfection agent, and a nucleic acid as described above. In some cases, the complex may also contain one or more additional peptides or proteins, eg, fusion-inducible, membrane permeable, intracellular internalization by transport or transport, or receptor-ligand peptides or proteins. These additional peptides or proteins may optionally be conjugated to a nucleic acid binding group, or may optionally be conjugated to a transfection agent (lipid or polycationic polymer), where the peptide Alternatively, the protein or modified peptide or protein is non-covalently associated with the nucleic acid. Without being bound by any theory, Applicants believe that the complexes of the present invention are typically lipid aggregates comprising liposomes or lipid aggregate structures. The exact nature of the structure is currently unknown. Thus, in certain exemplary embodiments, the complex of the present invention is a liposomal complex. The entire complex, or a portion of the complex, eg, a lipid moiety, eg, a lipid of formula I, can be formulated into liposomes using, for example, back evaporation methods known in the art. Alternatively, the lipid portion of the complex or the entire complex may be formulated by other well-known liposome forming methods such as sonication or microfluidization. These liposome formulations are effective for transfecting DNA into cultured cells.

  In one embodiment, a complex comprising a non-natural peptide or protein of the present invention and a nucleic acid (the non-natural peptide or protein can optionally be conjugated to a nucleic acid binding group) is first formed and then transferred. Combined with a cationic lipid, for example a lipid of formula I, for In related embodiments, conjugates of peptides or proteins and lipids are optionally combined with other lipids, including appropriate cationic lipids, and then combined with nucleic acids for transfection. In another related embodiment, the nucleic acid-lipid complex is formed and then combined with a non-natural peptide or protein for transfection. As described above, the lipid-containing complex of any of these embodiments can be a liposomal or non-liposomal formulation. Further, any of the complexes formed in these embodiments may be stored, for example, for 5 minutes to 1 year, or 15 minutes to 6 months, or 1 hour to 3 months prior to transfecting the cells. Can do. In the case of peptide or protein-lipid conjugates, such conjugates can be stored, for example, for 5 minutes to 1 year, or 15 minutes to 6 months, or 1 hour to 3 months prior to combining with the nucleic acid. it can.

  In another embodiment, after a complex containing a non-natural peptide or protein and a nucleic acid (the non-natural peptide or protein may be conjugated to a nucleic acid binding group) is formed for transfection. Combined with polycationic polymer. In a related embodiment, the peptide-polycationic polymer conjugate is combined with a nucleic acid for transfection, optionally combined with another polycationic polymer. In another related embodiment, the nucleic acid-polycationic polymer complex is formed and then combined with a peptide or protein for transfection. Polycationic polymers and / or peptide-conjugated polycationic polymers can be combined with cationic lipids and cationic lipid compositions to provide improved nucleic acid transfection compositions. According to the present invention, multiple peptides and / or proteins may be added to achieve transfection.

  Transfection compositions of the invention comprising a peptide or protein conjugate with lipid and nucleic acid may further comprise other non-peptide or non-protein agents known to further enhance transfection.

  The transfection composition of the present invention comprising a conjugate of a peptide or protein and a polycationic polymer and a nucleic acid may further comprise other non-peptide agents known to further enhance transfection of the polycationic polymer. For example, transfection of polycationic polymers can be enhanced by the addition of DEAE-dextran and / or chloroquine.

  In one preferred but non-limiting embodiment, a non-natural peptide of the invention can be first bound by non-covalent association to a nucleic acid or other cargo molecule to be introduced into the cell. . The peptide-nucleic acid complex is then mixed with a transfection agent (or mixture of agents) and the resulting mixture is used to transfect cells. Preferred transfection agents are cationic lipid compositions, such as, but not limited to, those comprising a lipid of formula (I), specifically monovalent and polyvalent cationic lipid compositions, more specifically cationic Consists of a 1: 1-4: 1 mixture of cationic lipids and DOPE and a 1: 1: 1-4: 1 mixture of cationic lipids and cholesterol, and a 1: 1-4: 1 mixture of cationic lipids and DOPC Cationic lipid composition, more specifically a 1: 1 to 4: 1 mixture of dihydroxyl-dimyristyl spermine tetrahydrochloride and DOPE and 1: 1 of dihydroxyl-dimyristyl spermine tetrahydrochloride and cholesterol ˜4: 1 mixture, as well as 1: 1 of dihydroxyl-dimyristylspermine tetrahydrochloride and DOPC. A 4: 1 mixture, and a 1: 1 to 4: 1 mixture of hydroxyl-dimyristyl spermine tetrahydrochloride and DOPE and a 1: 1 to 4: 1 mixture of hydroxyl-dimyristyl spermine tetrahydrochloride and cholesterol, and hydroxyl-di It is a cationic lipid composition composed of a 1: 1 to 4: 1 mixture of myristylspermine tetrahydrochloride and DOPC.

  In another optional embodiment, a fusion-inducing peptide or protein, a transport or transport peptide or protein, a receptor-ligand peptide or protein, or a nuclear internalization peptide or protein, and / or modified analogs thereof (eg, spermine A complex of one or more transfection-enhancing peptides, proteins, or protein fragments comprising a modified peptide or protein) and a non-natural peptide of the invention to be introduced into a cell; At the same time or shortly thereafter, it may be complexed with the nucleic acid. The peptide-nucleic acid complex is then mixed with a transfection agent and the resulting mixture is used to transfect cells. In certain embodiments, the mixture of transfection enhancing peptides, proteins, or protein fragments is stored prior to complexing with the nucleic acid.

  In another optional embodiment, the transfection agent component (lipid, neutral lipid, helper lipid, cationic lipid, dendrimer, or PEI) is directly or via a linking group or spacer group. It can be covalently conjugated to selected peptides, proteins, or protein fragments. Of particular interest in this embodiment are peptides or proteins that are non-natural fusion-inducing proteins derived from non-enveloped viruses, such as those known in the art.

Examples of use of complexes containing non-naturally occurring peptides of non-enveloped viruses Delivery methods using the lipid aggregates of the invention or mixtures thereof are in vitro, ex vivo, and in vivo, especially animal cells, human cells, non- It can be utilized for transfection into eukaryotic cells or tissues, including human animal cells, insect cells, plant cells, avian cells, fish cells, mammalian cells and the like. The polyanion to be delivered into the cell is contacted with the lipid aggregate in the presence of the non-natural peptide described above to form a polyanion-lipid-polypeptide aggregate complex. The target cell (s) are then incubated with the complex or, for in vivo applications, the complex is administered to the organism so that the complex contacts the target cell or tissue. The compounds of the present invention also have various useful molecules and substances, also referred to as transfection aids, such as proteins, peptides, to enhance cell targeting, uptake, internalization, nuclear targeting, and expression. It may be joined with a growth factor or the like, mixed with them, or used with them.

  The complexes and methods of the present invention, particularly those involving transfection compositions comprising the complexes provided herein, are more specifically disclosed for transfection of cells, particularly eukaryotic cells, in vitro and in vivo, and more specifically Can be used for transfection of higher eukaryotic cells, including animal cells. The methods of the invention can be used to generate transfected cells that express useful gene products. The methods of the invention can also be used as a step in the production of transgenic animals. The methods of the invention include gene therapy and viral inhibition methods, as well as antisense or antigene nucleic acids, ribozymes, RNA regulatory sequences, siRNA, RNAi, Stealth® RNAi (Invitrogen Corporation, Carlsbad Calif.), Or related inhibition. Can be useful as a step in any therapeutic method that requires the introduction of a nucleic acid into a cell, including a method for introducing a sex or regulatory nucleic acid into a cell. In particular, these methods may be useful in cancer therapy, in vivo and ex vivo gene therapy, and diagnostic methods.

  The transfection compositions and methods of the present invention comprising peptides, proteins, peptides or protein fragments, or modified peptides or modified proteins can also be used as research agents in any transfection of eukaryotic cells made for research purposes. Can be.

  Accordingly, provided herein is a method of introducing a macromolecule into a cell, the method comprising forming a transfection composition comprising a nucleic acid and a complex comprising a transfection agent and a fusion agent (a fusion agent). Includes amino acid sequences that facilitate fusion from non-enveloped viral fusion proteins), as well as contacting eukaryotic cells with the transfection composition. An exemplary protocol for transfection of eukaryotic cells using the compositions of the present invention is provided in the Examples section herein. As disclosed herein, the fusion agent in the illustrative example is a membrane fusion peptide (MPP), advantageously a fusion peptide that is 5-50 amino acids in length, wherein Wherein at least 5 consecutive amino acids of the fusion peptide are at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 to any of the peptides listed in Table 1. Or 100% similar.

  A further embodiment provides a method of transfecting a cell or tissue with a nucleic acid in vivo, the method comprising one or more cationic lipids, optionally one or more neutral lipids, and optionally 1 Conditions sufficient to form a lipid aggregate, preferably a liposome, comprising two or more helper lipids and to promote a stable non-feeding interaction between the peptide and the nucleic acid. Contacting the nucleic acid-peptide complex formed by contacting the nucleic acid with a non-natural peptide of the invention, thereby forming a neutral or positively charged lipid aggregate-nucleic acid complex; Administering the complex to an organism such that the lipid aggregate-nucleic acid complex contacts the target cell or tissue.

  Administration of the lipid aggregate-peptide-nucleic acid complex may be accomplished by oral, intravenous, subcutaneous or intramuscular injection, or may be applied topically to tissue or cells in culture in a laboratory environment.

  In some cases, the polyanion-peptide-lipid aggregate complex is stored for a period of time before being contacted with the cell (s) for transfection. The polyanion-peptide-lipid aggregate complex is stable and is at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 At least 20 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least It can be stored for 4 months, at least 5 months, at least 6 months, or at least 1 year, or any of these periods.

  In another embodiment, lipid aggregates of the present invention (approximately 1 μl to 2000 μl) are provided to the wells of a multi-well plate. A target polyanion molecule to be delivered to the target cell is selected and added to the well to form a polyanion-peptide-lipid aggregate complex that is subsequently contacted with the target cell. Lipid aggregates may have the same composition and concentration in each well, or the composition and / or concentration of lipid aggregates may vary from well to well. If the polyanion is a nucleic acid such as DNA or RNA, the nucleic acid may be added to the well and optionally stored prior to contacting the target cell.

  The methods of the present invention may optionally involve one or more cationic lipids and one or more helper neutral lipids prior to or simultaneously with contacting the nucleic acid-peptide complex with one or more cationic lipids. Contacting to form a lipid aggregate encapsulating the nucleic acid-peptide. The method also optionally includes forming lipid aggregates into liposomes prior to contacting with the nucleic acid. In further embodiments, the liposomes are formed by microfluidization, extrusion, or other means known in the art. The nucleic acid is preferably DNA or RNA that inhibits the expression of the target gene. Preferably, the nucleic acid associates with the transcript of the gene to effect inhibition. Preferably, the nucleic acid is RNAi, siRNA, shRNA, or stRNA, optionally chemically modified.

  The amount and concentration of a nucleic acid or other macromolecule, the amount and concentration of a transfection complex provided herein, the amount and concentration of a dilution, and the amount and concentration of a cell can be determined, for example, using a cytotoxicity assay. And / or standard experimental approaches for their optimization and titration, including methods using transfection using nucleic acid expression vectors that express reporter genes such as β-galactosidase, luciferase, and / or fluorescent proteins Can be determined using. In addition, cell density can be optimized using standard methods, and cell densities for transfection using the transfection complexes provided herein can be as high as, for example, greater than 75%. It can range from density to low density below 50%.

  Exemplary diluents for the complexation reaction include, for example, low serum or serum-free media such as D-MEM and RPMI 1640, and OptiPro ™, Opti-MEM® (Invitrogen Corporation) Is mentioned. Incubation times to form complexes can be determined using routine methods, but typical incubation times are 5 to 240 minutes. In addition, it will be appreciated that the medium for culturing cells before and after transfection may be selected based on the cell line undergoing transfection and based on the application of the particular method. For example, for protein production in suspension cells, in an exemplary embodiment, a low serum medium, or advantageously a serum free medium, may be used. In certain exemplary embodiments, animal origin-free media such as 293 expression media (Invitrogen Corporation) and CD-CHO media (Invitrogen Corporation) are used. In certain embodiments, depending on the cell line undergoing transfection, antibiotics can be excluded from the post-transfection medium. The incubation time for culturing the cells after transfection varies depending on the cell type and the desired transfection result, but typically ranges from 2 hours to days. For large scale protein production, the cells can be incubated as a non-limiting example, eg, 1-7 days.

  It will be appreciated that a wide range of concentrations of transfection agents and fusion agents can be used in the complexes, compositions, and methods provided herein. For example, in an exemplary non-limiting example of a composition comprising a complex comprising a cationic lipid and a non-natural peptide, the total non-limiting cationic lipid and untranslated peptide in the composition The combined concentration can be from 1 mg to 4 mg / ml. The range of peptides added to the lipid at 1 mg / ml can be from 100 μg / ml to 3 mg / ml. The ratio of cationic lipid to helper lipid can range from 0.5 / 1.0 (mole) to pure compound.

  Cells that can be transfected according to the present invention include virtually any eukaryotic cell, including, for example, primary cells, cells in culture, and cells in cultured tissue, particularly cells that appear difficult to transfect. Is included. The cells may be bound cells or cells in suspension. In certain exemplary embodiments, the cells are suspended CHO-S cells and suspended 293-F cells. Suspension cell cultures are particularly suitable for the protein production methods provided herein. Other cells that can be transfected using the agents and methods of the invention include 293, eg, GripTite 293 MSR (Invitrogen Corporation), CHO, Cos7, NIH3T3, Hela, primary fibroblasts, A549, Be2C, SW480, Caco2, primary neurons, Jurkat, C6, THP1, IMR90, HeLa, ChoK1, GT293, MCF7, HT1080, LnCap, HepG2, PC12, SKBR3, and K562 cells, or any cell listed in Table 6, It is not limited to these.

  In certain embodiments provided herein, the complex used to transfect a cell includes a transfection enhancing agent. For example, the transfection enhancing agent may be a nuclear internalization peptide. In one example, the transfection enhancing agent is PLUS ™ reagent (Invitrogen Corporation). The addition of PLUS ™ reagent has been shown to enhance protein expression when used with the transfection compositions provided herein. Cytotoxicity was not affected by the use of PLUS ™ reagent.

  In another embodiment, a method for producing a protein comprising transfecting a cell with a nucleic acid molecule encoding the protein, incubating the cell to produce the protein, and recovering the protein. Provided herein, where transfection is performed by contacting a cell with a transfection composition comprising a non-natural peptide of the invention. The composition for transfection of cells can be any composition provided herein. Exemplary compositions include a nucleic acid molecule encoding a protein of interest complexed with a non-natural peptide of the invention, optionally a fusion agent, and a transfection agent.

  In an exemplary embodiment, the encoded protein is an antibody molecule, or an antigen-binding fragment or derivative portion thereof, such as a single chain Fv fragment. In these embodiments, the method may further comprise isolating the protein by using affinity generation on an antibody binding column. In certain examples, nucleic acids encoding both chains of the antibody are transfected into cells using the transfection compositions provided herein.

  It will be appreciated that the nucleic acid encoding the protein may be an expression vector. An expression vector typically has a promoter operably linked to one or more nucleic acids encoding one or more protein chains. If the protein produced is a pharmaceutical product, the protein can be formulated as appropriate, for example, in an appropriately chosen physiological medium.

  The transfection compositions provided herein can also be used to introduce peptides and proteins into cells using methods known in the art. Methods of using cationic lipids for peptide and protein delivery have already been described. In addition, transfection compositions can be used to deliver nucleic acids, peptides, and proteins into tissues in vivo. Methods for using lipids to deliver compounds to tissues in vivo have already been described. The transfection composition can also be used for therapeutic and diagnostic applications using appropriately selected physiological media.

Reagent kit:
The present invention is further directed to a kit comprising in at least one first suitable container at least one non-natural peptide according to the present invention. The kit of the present invention further comprises one or more containers comprising a reagent that facilitates the introduction of at least one macromolecule, such as a cationic transfection reagent, optionally one or more helper lipids or neutral lipids. In addition, in some cases, cargo may be provided, for example nucleic acids or other cargo as defined above. Preferred transfection reagents include, but are not limited to, cationic lipids.

  The components of the transfection composition of the present invention can be provided in a reagent kit. The kit can include a transfection agent and a non-natural peptide of the invention. The kit may also optionally include a transfection enhancing agent, such as a transfection enhancing peptide, protein, or fragment thereof, or a transfection enhancing compound. Transfection agents, non-natural peptides, and optional transfection enhancers, if present, may each be included as a mixture (ie, in a single container, typically a tube and / or vial). Or alternatively as separate parts (ie in separate containers, eg separate vials and / or tubes). As will be appreciated, the kit of the present invention typically includes a container, such as a vial and / or tube, which is packaged together, for example, in a cardboard box or other packaging container. . The kit can be shipped from the supplier to the customer. For example, in one embodiment, provided herein is a kit comprising a vial comprising a liposomal formulation comprising a transfection agent and a transfection enhancing peptide. The kit may also include a separate container containing a transfection-enhancing agent such as, for example, a transfection-enhancing peptide, such as Plus Reagent ™ (Invitrogen Corp., Carlsbad, Calif.). The kit may also include cells, cell culture medium, and a reporter nucleic acid sequence, eg, a plasmid expressing the reporter gene, in separate containers. In certain examples, the culture medium can be a low serum medium and / or a protein expression medium.

  In one embodiment, the kit is a cationic lipid, such as, but not limited to, a lipid of formula I, optionally in combination with one or more helper lipids and / or one or more neutral lipids, and the present Inventive peptides, proteins, or fragments thereof, or individual portions of modified peptides, proteins, or fragments thereof, or mixtures thereof. In another embodiment, the kit comprises a polycationic polymer and an inventive peptide, protein, or fragment thereof, or individual portions of a modified peptide, protein, or fragment thereof, or mixtures thereof. The cationic lipid transfection kit optionally includes neutral lipids, as well as other transfection enhancers or other additives, so that the relative amounts of the components included in the kit may facilitate the preparation of the transfection composition. Can be adjusted to. Kit components may include appropriate media or solvents for other kit components.

  Cationic lipid transfection kits comprising monocationic or polycationic lipid compositions, such as but not limited to lipids of formula I, further comprising neutral lipids and peptides or proteins of the invention are preferred.

  The dendrimer transfection kit may optionally include other transfection enhancing agents such as DEAE-dextran and / or chloroquine, and other additives, and the relative amounts of the components included in the kit may vary depending on the transfection composition. Can be adjusted to facilitate preparation.

  The kit provided by the present invention includes a polycationic lipid composition comprising DOSPA and DOPE, or an individual portion of a monocationic lipid composition comprising DOTMA and DOPE, and a modified peptide, optionally spermine or spermidine modification. And those containing a part of the peptide. Kits provided by the present invention include those comprising individual portions of a polycationic polymer and a portion of a spermine modified peptide.

  In a related embodiment, the kit of the invention comprises a conjugate of a peptide or protein and a lipid or a conjugate of a peptide or protein and a polycationic polymer to a non-conjugated lipid, non-conjugated poly-type that facilitates transfection Cationic polymers and other drug combinations may be included.

  Kits of the invention can include those useful in diagnostic methods, such as diagnostic kits, which include transfection agents and transfection enhancing agents (eg, proteins, peptides, and peptide and protein fragments and In addition to (modified forms), diagnostic nucleic acids may be included. Diagnostic nucleic acid is a general term for any nucleic acid that can be used to detect the presence of another substance (most commonly an analyte) in a cell. For example, when transfected into a cell, a diagnostic nucleic acid can respond to the presence of another substance (eg, a protein, small molecule, steroid, hormone, or another nucleic acid) in the cell in order to express the gene there. May be increased or decreased. A diagnostic nucleic acid also includes a nucleic acid with some label for detection of a target cell or tissue or for detection of a substance in the target cell or tissue or otherwise with a detectable marker for a particular target cell or tissue. included.

  Nucleic acids that can be transfected by the methods of the present invention include DNA and RNA of any size derived from any source, including natural or non-natural bases, and for therapeutic or other uses. One that encodes a useful protein and one that can express it in a cell, one that inhibits the expression of an undesirable nucleic acid in the cell, one that inhibits an undesirable enzyme activity or activates a desired enzyme, Those that catalyze the reaction (ribozymes) and those that play a role in diagnostic assays (eg, diagnostic nucleic acids). Therapeutic nucleic acids include those that encode or express intracellularly useful proteins, peptides, or polypeptides, those that inhibit the expression of unwanted nucleic acids in cells, and intracellular In which undesirable enzyme activity is inhibited or which activates the desired enzyme.

  The compositions and methods provided herein also provide for biologically active macromolecules other than nucleic acids, including polyamines, polyamic acids, polypeptides, and proteins, among others, in light of the disclosure herein. It can be easily adapted for introduction into eukaryotic cells. Other materials useful as therapeutic agents, diagnostic materials, research reagents, for example, that can be conjugated to peptides and modified peptides and introduced into eukaryotic cells by the methods of the invention.

  The lipid of Formula I can be used as the cationic lipid (s) in the kit described above, which can be independently provided in a reagent kit. In general, the kit contains a lipid of formula (I) in a suitable container. The lipid may be, for example, in a solution of an organic solvent, such as in ethanol, in a buffer, or in a solvent / buffer mixture. In addition, the kit includes a lipid of formula (I), as well as Amino acid sequences derived from non-natural proteins that enhance or facilitate membrane fusion between the liposome carrier and the cell membrane in a suitable solvent or buffer can be included, but are not limited to these.

  In one embodiment, the kit comprises, for example, a lipid of formula (I) or other cationic lipid and a peptide, protein, or fragment thereof, or a separate part of a modified peptide, protein, or fragment thereof, or a mixture thereof Can be included. Kits containing lipids of formula (I) or other cationic lipids may optionally contain neutral lipids, as well as other transfection enhancing agents or other additives, and the relative amounts of components in the kit Can be adjusted to facilitate the preparation of the transfection composition. Kit components may include appropriate media or solvents for other kit components.

  Preferred are kits comprising lipids of formula (I) or other cationic lipids, neutral lipids, and modified peptides or proteins. The kit provided by the present invention includes individual parts of the lipids of formula (I), DOPE, and parts of peptides (particularly spermine-modified peptides). The kits provided by the present invention include individual portions of the lipids of formula (I) as well as portions of modified peptides containing a range of basic amino acids such as lysine, ornithine, or arginine.

Methods of Sale Also provided are methods for selling the non-natural peptides, lipids, transfection complexes, transfection compositions, and / or kits provided herein, including: Presenting an identifier identifying the non-natural peptide, lipid, complex, and / or transfection composition, and / or kit provided to the customer, and the non-natural peptide provided herein, It includes providing customers with access to purchasing functions for purchasing lipids, transfection complexes, transfection compositions, and / or kits. The identifier is typically presented to the customer as part of the ordering system. The ordering system may include an input function for identifying the desired product and a purchase function for purchasing the identified desired product. The ordering system is typically under direct or indirect control of the provider. As used herein, a customer refers to any individual, institution, company, university, or organization that seeks to obtain products and services for biological research. As used herein, a provider refers to any individual, institution, company, university, or organization that seeks to provide products and services for biological research.

  The present invention also provides a method for selling the non-natural peptides, lipids, transfection complexes, transfection compositions, and / or kits provided herein, including a telephone ordering system. Presenting the input function of the user to the customer and / or presenting the customer with a list of data entry fields or selectable entries as part of the computer system, where non-natural peptides, lipids, The injection complex, transfection composition, and / or kit are identified using the input function. If the input function is part of a computer system, such as displayed on one or more pages of an Internet site, the customer is typically presented with an online purchase function, such as an online shopping cart, where: The purchase function is used to purchase customer-identified non-natural peptides, lipids, transfection complexes, transfection compositions, and / or kits. In one aspect, different non-natural peptides, lipids, complexes, and / or transfection compositions and / or kits provided herein are identified, or different amounts or weights of the present specification. The customer is provided with a plurality of identifiers that identify the non-natural peptides, lipids, complexes, and / or transfection compositions and / or kits provided in the documentation. The method may further comprise enabling a purchasing function for purchasing the lipids, transfection complexes, transfection compositions, and / or kits provided herein. The method may still further comprise shipping purchased non-natural peptides, lipids, transfection complexes, transfection compositions, and / or kits provided herein to the customer. Non-natural peptides, lipids, transfection complexes, transfection compositions, and / or kits can be shipped from the provider to the customer. The provider typically controls input functions and inputs to purchase non-natural peptides, lipids, complexes, and / or transfection compositions and / or kits provided herein. You can control the websites that are accessed to access features.

Pharmaceutical Compositions The transfection agents and transfection enhancing agents of the present invention can be provided in various pharmaceutical compositions and dosage forms for therapeutic use. For example, injectable preparations, nasal preparations, and preparations for intravenous and / or intralesional administration containing these complexes can be used for therapy.

  In general, the pharmaceutical compositions of the invention provide for the introduction of a sufficiently high level of nucleic acid into a target cell or tissue such that the nucleic acid has the desired therapeutic effect in the target cell or tissue. Should contain sufficient transfection agent and any enhancer (peptide, protein, etc.). The level of nucleic acid in the target cell or tissue that will be effective for treatment will depend on the effectiveness of the inhibition or other biological function as well as the number of sites to which the nucleic acid should affect.

  The dosage at which a transfection composition described herein is administered to a patient will depend on a number of other factors, including the method and site of administration, the patient's age, weight, and condition. One skilled in the art can readily adjust the dosage for a given dosage type, a given patient, and a given therapeutic application.

  Containing an inhibitory element, such as serum or high salt levels, where the transfection composition may inhibit the introduction of nucleic acid into the cell or otherwise interfere with transfection or nucleic acid complex formation One skilled in the art will appreciate that the amount must be minimal. Any pharmaceutical or therapeutic composition must have a minimum content of components that can cause adverse side effects to the patient, depending on the specific application.

  The transfection compositions described herein can be formulated into a composition comprising a pharmaceutically active agent and a pharmaceutically acceptable diluent, excipient, or carrier therefor. Such compositions may be tablets, pills for oral, parenteral (eg, intravenous, intramuscular, or subcutaneous), nasal, sublingual, or rectal administration, or administration by inhalation or insufflation. , Capsules (including sustained or delayed release formulations), powders, granules, elixirs, tinctures, syrups and emulsions, sterile parenteral solutions or suspensions, aerosols or liquid sprays, drops, ampoules, automatic infusion devices, Or in unit dosage forms, such as suppositories, incorporated herein by reference, in a suitable manner, and by Remington's Pharmaceutical Sciences, (Gennaro, ed., Mack Publishing Co., Easton Pa., 1990). To be disclosed, etc.) By convention to have, it may be formulated.

  Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline Examples include cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl- and propyl-hydroxybenzoate, talc, magnesium stearate, and mineral oil. The formulations may further include lubricants, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents, or flavoring agents. Where the carrier serves as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for the active ingredient. For injection, it is possible to prepare a solution of one or more lipids of the invention in a pharmaceutically acceptable carrier, such as an aqueous or non-aqueous solvent. Examples of solvents that can be used are distilled water for injection, physiological saline, Ringer's solution, vegetable oil, synthetic fatty acid glycerides, higher fatty acid esters, propylene glycol and the like.

  The following examples are provided to illustrate certain embodiments of the present disclosure and to assist one of ordinary skill in practicing the present disclosure. These examples are in no way considered to limit the scope of the present disclosure.

Example 1. Lipid Aggregate / Polypeptide Complex Preparation and Cultured Cell Transfection All cells were cultured under standard culture conditions for each cell line recommended by the American Type Culture Collection (ATCC). Approximately 24 hours prior to transfection, cells were seeded so that the cells were 70-90% confluent on the day of transfection. In general, the following guidelines are applicable, but as will be readily appreciated by those skilled in the art, depending on the identity of the cell line, its growth characteristics and requirements, and its morphology in the adherent culture, There are major changes. In general, for 96-well plates, 1-4 × 10 ⁴ cells are seeded per well, and for 24-well plates, 0.5-2 × 10 ⁵ cells are seeded per well, 6 wells For plates, 0.25 to 1 × 10 ⁶ cells were seeded.

  Transfection complexes for transfecting DNA into cells in culture were prepared according to the manufacturer's protocol. LIPOFECTAMINE (registered trademark) 2000 and LIPIFECTAMINE (registered trademark) LTX are available from Life Technologies Corp. (Carlsbad, CA), FUGENE® HD is available from Promega Corp. (Fitchburg, WI) and X-TREMEGENE ™ HP was purchased from Roche Diagnostics (Basel, Switzerland).

を有するペプチド１を合成し、乾燥粉末として得た。乾燥粉末を滅菌超純水中で、４．３５ｍｇ／ｍｌの最終濃度に再構成し、完全に溶解させた。このストックペプチド溶液を次のステップで使用するために取り置いた。 To prepare a transfection complex containing the non-natural peptide described above, the following sequence:

Peptide 1 having was synthesized and obtained as a dry powder. The dry powder was reconstituted in sterile ultrapure water to a final concentration of 4.35 mg / ml and dissolved completely. This stock peptide solution was set aside for use in the next step.

  To prepare lipid aggregate-DNA-peptide complexes, LIPOFECTAMINE® 3000 reagent (Life Technologies Corp., Carlsbad, Calif.) Was obtained. For cells in each well of a 96-well, 24-well, or 6-well plate to be transfected, 5 μl, 25 μl, and 125 μl aliquots of Gibco® Opti-MEM® medium are placed in separate disposables. In a plastic Eppendorf tube. 0.1 μl to 0.6 μl for 96 well plate, 0.5 μl to 3.0 μl for 24 well plate, or 2.0 μl to 15 μl for 6 well plate, LIPOFECTAMINE® 3000 reagent aliquoted Opti -Added to MEM (R), mixed well and incubated at room temperature.

  In separate eppendorf tubes, 5 μl (for each well of a 96-well plate), 25 μl (for each well of a 24-well plate), or 125 μl (for each well of a 6-well plate) Gibco® Opti-MEM (registered) The medium was aliquoted into tubes for each well of cultured cells to be transfected, where 0.1 μg pcDNAEF1a / emGFP or GST-STAT expression vector DNA, 24 well plate for each well of a 96 well plate 0.5 μg pcDNAEF1a / emGFP or GST-STAT expression vector DNA for each well and 2.5 μg pcDNAEF1a / emGFP or GST-STAT for each well of a 6-well plate It was mixed with the current vector DNA.

  To the diluted DNA mixture, add 0.2 μl stock peptide for each well of a 96-well plate, 1 μl stock peptide for each well of a 24-well plate, 5 μl stock peptide for each well of a 6-well plate, The peptide / DNA mixture was mixed well and incubated at room temperature for approximately 1 minute.

  For each well of a 96-well plate, 5 μl of the diluted DNA / peptide mixture is mixed with 5 μl of diluted LIPOFECTAMINE® 3000 reagent, and for each well of a 24-well plate, 25 μl of the diluted DNA / peptide mixture is mixed with 25 μl Mix with diluted LIPOFECTAMINE® 3000 reagent, and for each well of a 6-well plate, mix 125 μl of diluted DNA / peptide mixture with 125 μl of diluted LIPOFECTAMINE® 3000 reagent and mix the resulting mixture with A lipid-peptide-DNA complex was formed by incubating at room temperature for approximately 5 minutes.

  After incubation, the lipid-peptide-DNA complex was added to the cells that had been seeded the previous day using fresh growth medium; for 96-well plates, 10 μl lipid-peptide-DNA was added to the cells; For 24-well plates, 50 μl of lipid-peptide-DNA mixture was added to the cells, and for 6-well plates, 250 μl of lipid-peptide-DNA was added to the cells. Cells were incubated for approximately 24-48 hours in the presence of lipid-peptide-DNA and analyzed.

Example 2 Transfection of various cell lines A panel of 10 cancer cell lines (HepG2, Hepa1-6, Hep3B, HUH7, MCF-7, MDA-MB-23, SKBR3, LNCaP, Bend3, and T986) that are difficult to transfect Two neuronal cell lines that are difficult to transfect (PC12 and Neuro2A), two myoblast cell lines that are difficult to transfect (H9C2 and C2C12), and one renal fibroblast cell line that is difficult to transfect (Vero) According to the method described in Example 1, pcDNAEF1a / emGFP which is an expression vector code GFP was transfected. Cells were visualized using a fluorescence microscope at the appropriate wavelength after 24 hours in the presence of the transfection complex.

  The expression of GFP in the cancer cell line is shown in FIG. 1A, the one in nerve cells is shown in FIG. 1B, the one in myoblasts is shown in FIG. 1C, and the one in kidney fibroblasts is shown in FIG. 1D.

Example 3 Comparison of various transfection reagents Panels of 6 different cell lines (HEK293, HeLa, COS-7, LNCaP, A549, and HepG2) include FUGENE® HD, LIPOFECTAMINE® 2000, or Example 1 PcDNAEF1a / emGFP was transfected using LIPOFECTAMINE® 3000 in combination with peptide 1 described in 1. Cells were transfected for 48 hours. The results shown in FIG. 2 show that both FUGENE® HD and LIPOFECTAMINR® 2000 were able to transfect a small portion of each cell (FIG. 2, first two rows), but the transfection complex It shows that the presence of peptide 1 in the transfection efficiency was substantially improved (FIG. 2, last row).

To expand this study, a panel of 61 different cell lines was described above using either LIPOFECTAMINE® 2000 or LIPOFECTAMINE® 3000 in combination with peptide 1 described in Example 1. Transfected as follows. Approximately 48 hours after transfection, transfected cells are examined by fluorescence microscopy to determine relative transfection efficiency, cell extracts are prepared and analyzed using a FL600 fluorescent microplate reader A fold improvement in GFP expression over LIPOFECTAMINE® 2000 with LIPOFECTAMINE® 3000 with 1 was measured. The results are shown in Table 6. As can be seen, the use of LIPOFECTAMINE® 3000 reagent in combination with peptide 1 results in higher transfection efficiency and protein expression than LIPOFECTAMINE® 2000 reagent when tested in various cell lines. can get.

Example 4 Effect of transfection reagent dosage on transfection efficiency and protein expression HeLa cells are seeded in 96-well plates and 0.1 μl, 0.2 μl, 0.3 μl, or 0.4 μl of LIPOFECTAMINE® 2000 LTX or PcDNAEF1a / emGFP was transfected using LIPOFECTAMINE® 3000 in combination with peptide 1 described in Example 1. Transfection efficiency as measured by relative luminescence and relative protein expression were determined for each condition. The results are shown in FIG.

  FIG. 3A shows LIPOFECTAMINE® 2000 (open circles), LIPOFECTAMINE® LTX (open squares), and LIPOFECTAMINE combined with peptides according to one embodiment (increase doses), three different commercially available lipid aggregate formulations. FIG. 6 is a graph comparing the relative transfection efficiencies of expression vectors encoding GFP transfected into cultured HeLa cells using registered trademark 3000 (white triangles). The presence of peptide 1 in the transfection complex improves the transfection efficiency of the cells over the full range of transfection reagent doses tested. The improvement in transfection efficiency is particularly noticeable at the lowest dose tested.

  FIG. 3B shows LIPOFECTAMINE® 2000 (open circles), LIPOFECTAMINE® LTX (open squares), and LIPOFECTAMINE combined with peptides according to one embodiment, in increasing doses of three different commercially available lipid aggregate formulations ( It is the graph which compared the intensity | strength of the GFP expression in the HeLa cell which transfected the expression vector which codes GFP using a registered trademark 3000 (white triangle). The presence of peptide 1 in the transfection complex improves the relative expression of GFP in the cells over the entire range of transfection reagent doses tested. The improvement in protein expression is particularly noticeable at the lowest dose tested.

Example 5. Improved protein expression compared to three commercially available transfection reagents LIPOFECTAMINE® 2000, FUGENE® HD, X-TREMEGENE ™ HP, or as described in Example 1 LEPOFECTAMINE® 3000 in combination with Peptide 1 was used to transfect expression vectors encoding GST-STAT fusion proteins into HepG2 cells in 24-well plates. Approximately 24 hours after transfection, a cell lysate was prepared using NOVEX® Cell Extraction Buffer (Life Technologies, Carlsbad, Calif.), And the lysate was resolved by SDS-PAGE electrophoresis, It was transferred to a PVDF membrane, immunoblotted with an anti-GST HRP labeled polyclonal antibody, and detected with Pierce ™ ECL Western Blotting Substrate (Pierce Biotechnology, Rockford, IL).

  FIG. 4 is a Western blot comparing GST-STAT fusion protein (upper panel) in HepG2 cells transfected with an expression vector encoding GST-STAT fusion protein using the following commercially available lipid aggregate formulation: LIPOFECTAMINE (Registered trademark) 2000 (first lane), LIPOFECTAMINE® 3000 (second lane), FUGENE® HD (third lane), and X-TREMEGENE combined with a peptide according to one embodiment. TM HP (last lane). The lower panel shows a western blot of endogenous β-actin to confirm equal cytoplasmic extract loading in each lane.

Example 6 Transfection of H9 human embryonic stem cells H9 human embryonic stem cells were seeded at a density of 37500 cells / well in each well of a 96 well plate and 0.1 μl to 0.6 μl of LIPOFECTAMINE® 2000 or Example 1 50 μg, 100 μg, or 200 μg of pcDNAEF1a / emGFP was transfected with any of LIPOFECTAMINE® 3000 in combination with peptide 1 described in 1. Transfection efficiency was determined 24 hours after transfection.

  FIG. 5A shows increasing doses using either 0.1-0.6 μl per well of LIPOFECTAMINE® 2000 (open triangle) or LIPOFECTAMINE® 3000 in combination with a peptide according to one embodiment. Relative transfection efficiency of H9 human embryonic stem cell line (37,500 cells per well of 96 well plate) transfected with a GFP expression vector (50 μg left panel, 100 μg center panel, 200 μg right panel) It is a graph to compare.

  FIG. 5B shows 200 μl LIPOFECTAMINE® 2000 (left panel, showing 18% H9 cell transfection efficiency) or LIPOFECTAMINE® 3000 (right panel, 52% combined with peptide according to one embodiment). Is a representative fluorescent image of GFP expression in H9 cells cultured in 96-well plates, transfected with 100 μg / well using any of

Example 7 Genomic modification of cells using the CRISPR nuclease vector system Plasmid design and preparation: GENEART® Precision TALs and GENEART® CRISPR nuclease vectors were transferred to Life Technologies GENART® web design tools / lifetechnologies / communities.com. us / en / home / life-science / cloning / gene-synthesis / genele-precise-tals.html). The forward and reverse TALENs contain FokI nuclease and target the safe harbor locus of AAVS1. The fully integrated CRISPR vector system contains a Cas9 nuclease expression cassette and a guide RNA cloning cassette that target the safe harbor locus of AAVS1 in combination with a downstream orange fluorescent protein (OFP) reporter. A negative control plasmid, PCDNA ™ 3.3, was also used throughout the assay. The plasmid was transformed into competent E. coli cells. Clones were purified using the PURELINK® HiPure Plasmid Filter Maxiprep Kit after analysis and sequencing for specificity to ensure low endotoxin activity and high quality DNA.

U2OS and HepG2 cells were cultured for 4-5 passages in high glucose GIBCO® DMEM with GLUTAMAX ™ supplement and 10% fetal calf serum after thawing and the cells were dissociated with TRYPLE ™ Express. The enzyme was dissociated and seeded in 1 mL complete medium at 2 × 10 ⁵ cells per well in a 12-well plate to ensure 70-90% confluence on the day of transfection.

  LIPOFECTAMINE® 3000 reagent and LIPOFECTAMINE® 2000 reagent combined with peptide 1 were compared for each cell type. For transfection with LIPOFECTAMINE® 3000 reagent, in separate tubes, 1.5 μL LIPOFECTAMINE® 3000 reagent and 1 μg plasmid DNA, respectively, 50 μL OPTI-MEM® low serum medium After dilution in, 2 μL of peptide 1 (see Example 1) was added to the diluted DNA. Diluted DNA with peptide 1 was added to LIPOFECTAMINE® 3000 reagent and incubated for 5 minutes at room temperature. The resulting complex of 100 μL was then added to the cells in complete medium. The procedure was the same for the LIPOFECTAMINE® 2000 reagent, but the transfection reagent was increased to 3 μL and peptide 1 was added to the diluted DNA before adding it to the diluted LIPOFECTAMINE® 2000 reagent. Except that is added. All downstream analysis was performed 72 hours after transfection.

  OFP expression from the CRISPR vector was determined by flow cytometry and microscopic analysis. Images with an RFP filter were acquired using EVOS® FL Imaging System. Then 72 hours after transfection, they were dissociated using TRYPLE ™ Express enzyme and analyzed using a BD ACCURI ™ C6 flow cytometer with FL-2 filter and blue laser.

  FIG. 6 shows transfection efficiency and protein expression using CRISPR vectors in U2OS cells (FIG. 6A) and HepG2 cells (FIG. 6B). 6B). The vector that contained the OFP reporter gene was transfected with LIPOFECTAMINE® 2000 or LIPOFECTAMINE® 3000 reagent in combination with peptide 1. The bar graph shows relative OFP gene expression (measured by fluorescence intensity) and the fluorescence image below the bar graph shows the quantified fluorescence intensity of the corresponding OFP-expressing cells.

Genomic Cleavage Detection: The GENEART® Genomic Cleavage Detection Kit provides a reliable and rapid method for detection of locus-specific cleavage. Transfected cells were dissociated with TRYPLE ™ Express, washed with Dulbecco's phosphate buffered saline, and pelleted by centrifugation. Cells were lysed with the cell lysis buffer and proteolytic mix of the GENEART® Genomic Cleavage Detection Kit. After DNA extraction, PCR amplification was performed with forward and reverse primers. A denaturation and reannealing reaction was then performed to randomly anneal the mutated and unmutated PCR fragments. Detection enzyme (1 μL) was added and the mix was incubated at 37 ° C. for 1 hour, then the entire mix was electrophoresed on an E-Gel® EX 2% agarose gel to determine the percentage of genomic modification. Using ALPHAVIEW (TM) software, the cleavage efficiency was determined using the following formula: cleavage efficiency = 1-[(1-cleaved portion) ^1/2 ].

  FIG. 7A shows cleavage of TALEN and CRISPR targeting the AAVS1 locus in U2OS cells using either LIPOFECTAMINE® 2000 or LIPOFECTAMINE® 3000 in combination with Peptide 1 according to one embodiment. Shows efficiency.

  FIG. 7B shows cleavage of TALEN and CRISPR targeting the AAVS1 locus in HepG2 cells using either LIPOFECTAMINE® 2000 or LIPOFECTAMINE® 3000 in combination with peptide 1 according to one embodiment. Shows efficiency.

  Conclusion: U2OS cells derived from human osteosarcoma and HepG2 cells derived from human hepatocellular carcinoma were transfected with LIPOFECTAMINE® 3000 and peptide 1. Both cell lines showed improved transfection efficiency and protein expression compared to LIPOFECTAMINE® 2000 mediated transfection. Transfection efficiency and protein expression were assessed using a CRISPR construct containing an OFP reporter gene. U2OS cells transfected with LIPOFECTAMINE® 3000 and peptide 1 showed a 2-fold improved transfection efficiency (data not shown) and a 4-fold improved fluorescence intensity (FIG. 6A). HepG2 cells showed a 20-fold improvement in transfection efficiency (data not shown) and an 80-fold higher fluorescence intensity (FIG. 6B). Remarkably, an increase in cleavage mediated by TALEN and CRISPR was confirmed for the AAVS1 target locus in both LIPOFECTAMINE® 3000 and peptide-transfected cells, increased transfection, and implicitly It shows that protein expression will increase the cleavage rate of TALEN and CRISPR. U2OS cells transfected with LIPOFECTAMINE® 3000 and peptide 1 showed a 1.5-fold improved TALEN cleavage efficiency and a slightly improved CRISPR cleavage (FIG. 7A). HepG2 cells had a 3-fold higher cleavage efficiency for TALEN and an 8-fold higher cleavage efficiency for CRISPR (FIG. 7B).

Example 8 FIG. Lipid transfection reagents and peptides are required to enhance transfection HeLa cells are seeded in 96-well plates and 0.05 μl, 0.1 μl, 0.2 μl, 0.3 μl, 0.4 μl, or 0 5 μl of LIPOFECTAMINE® 3000 reagent alone (LF3K), LIPOFECTAMINE® 2000 (LF2K), peptide 1 alone (peptide 1), or LIPOFECTAMINE® in combination with peptide 1 described in Example 1 ) 3000 (LF3K + Peptide 1) were used to transfect 0.2 μg / well pcDNAEF1a / emGFP for 48 hours. Transfection efficiency and protein expression measured by fluorescence intensity (FL1-H) were determined. The results are shown in FIG.

  FIG. 8 shows LIPOFECTAMINE® 3000 alone (LF3K), LIPOFECTAMINE® 2000 (LF2K), peptide according to one embodiment (p4), or combination with peptide according to one embodiment, at the indicated doses (μl). LIPOFECTAMINE® 3000 (LF3K + peptide) was used to compare the relative transfection efficiency of HeLa cells transfected with the GFP expression vector (top graph, GFP + as% of single cells only) or relative per cell Two bar graphs representing GFP expression levels (bottom graph, average FL1-H of single cells only) are shown. The presence of peptide 1 in a cationic lipid aggregate formulation (eg, LIPOFECTAMINE® 3000) significantly enhances transfection efficiency and protein expression.

を有する；ペプチドＢ（本発明の非天然のペプチドのリンカー領域に対応）であり、ペプチド配列

を有する；ペプチドＣ（本発明の非天然のペプチドのカチオン性領域に対応）であり、ＣＰ１

のペプチド配列を有する；ペプチドＤ（本発明の非天然のペプチドのカチオン性領域に融合されたリンカー領域に対応）であり、ペプチド配列

を有する；及びペプチドＥ、ペプチド１に対応し、配列

を有する。 Example 9 Full length peptides are required for optimal transfection The following peptides are shown schematically in FIGS. 9A, 10A, and 11A and were synthesized as described in Example 1 for peptide 1 and purified Dissolved in water. Peptide A (corresponding to the MPP region of the non-natural peptide of the present invention), the peptide sequence

A peptide B (corresponding to the linker region of the non-natural peptide of the present invention), the peptide sequence

Peptide C (corresponding to the cationic region of the non-natural peptide of the invention), CP1

Peptide D (corresponding to the linker region fused to the cationic region of the non-natural peptide of the present invention), and the peptide sequence

And corresponding to peptide E, peptide 1 and the sequence

Have

  HepG2, A549, and MDA-MB-231 cells are seeded in a 24-well plate and peptide A, peptide B, peptide C, peptide D, peptide A and B together, peptide A and C together, peptide LIPOFECTAMINE® 3000 reagent in combination with A and D, peptide B and C, or in combination with peptide E, or peptide A, peptide B, peptide C, peptide D, without lipid transfection agent 1 μg using either peptide A and B together, peptide A and C together, peptide A and D together, peptide B and C together, or peptide E alone Of pcDNAEF1a / emGFP was transfected for 48 hours. Cells were visualized using fluorescence microscopy and transfection efficiency as measured by percentage of GFP + cells and protein expression as measured by average fluorescence per cell was determined as described above. The results are summarized in FIGS. 9B, 9C, 10B, 10C, 11B, and 11C.

  FIG. 9B detects GFP expression in HepG2 cells transfected with an expression vector encoding GFP using LIPOFECTAMINE® 3000 in the presence of the indicated peptide or peptide combination (shown in FIG. 9A). To represent a series of fluorescent images.

  FIG. 9C shows the transduction of an expression vector encoding GFP using LIPOFECTAMINE® 3000 in the presence of one of the indicated peptides A to E or the possible peptide combinations (shown in FIG. 9A). Two bar graphs showing mean fluorescence per cell (top graph) and transfection efficiency (% GFP + cells) in the transfected HepG2.

  FIG. 10A represents a peptide map of the various peptides or peptide fragments used in the experiments in A549 cells shown in FIGS. 10B and 10C, where peptide A is MPP peptide alone and peptide B is linker peptide alone. Peptide C is a cationic peptide alone, peptide D is a linker peptide fused to a cationic peptide, and peptide E is a full-length peptide in which peptide A is fused to peptide D.

  FIG. 10B shows the expression of GFP in cultured A549 cells transfected with an expression vector encoding GFP transfected with LIPOFECTAMINE® 3000 in the presence of the indicated peptide or peptide combination (shown in FIG. 10A). Represents a series of fluorescent images to detect.

  FIG. 10C shows transduction of an expression vector encoding GFP using LIPOFECTAMINE® 3000 in the presence of one of the indicated peptides A to E or the indicated peptide combination (shown in FIG. 10A). 2 represents two bar graphs showing average fluorescence per cell (top graph) and transfection efficiency (% GFP + cells) in infected A549 cells.

  FIG. 11A represents a peptide map of the various peptides or peptide fragments used in the experiments in the MDA-MB-231 cells shown in FIGS. 11B and 11C, where peptide A is the MPP peptide alone and peptide B is A linker peptide alone, a peptide C is a cationic peptide alone, a peptide D is a fusion of a linker peptide to a cationic peptide, and a peptide E is a full-length peptide in which a peptide A is fused to a peptide D. is there.

  FIG. 11B shows cultured MDA-MB-231 cells transfected with an expression vector encoding GFP transfected with LIPOFECTAMINE® 3000 in the presence of the indicated peptide or peptide combination (shown in FIG. 11A). FIG. 6 represents a series of fluorescent images that detect GFP expression in.

  FIG. 11C shows the transduction of an expression vector encoding GFP using LIPOFECTAMINE® 3000 in the presence of one of the indicated peptides A to E or the possible peptide combinations (shown in FIG. 11A). Two bar graphs showing mean fluorescence per cell (upper graph) and transfection efficiency (% GFP + cells) in the infected MDA-MB-231.

  It will be appreciated that other suitable modifications and adaptations to the methods and applications described herein will be apparent and may be made without departing from the scope of the invention or any embodiment thereof. Will be readily apparent to those skilled in the art. Having described the invention in detail, the invention will be more clearly understood by reference to the following examples, which are included herein for purposes of illustration only and are intended to limit the invention. Is not intended.

  All publications, patents, and patent applications mentioned in this specification are intended to indicate that each individual publication, patent, or patent application is specifically and individually incorporated herein by reference. To the same extent, are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.

Claims (32)

Construction

A peptide having the formula:
A is a membrane permeable peptide,
L is a bond or linker peptide;
B wherein said peptide is a cationic moiety or a cationic polypeptide.   2. The peptide of claim 1, wherein A comprises a peptide sequence selected from Table 1.   The peptide according to claim 1, characterized in that A is a peptide comprising 5 to about 50 amino acids, and A improves the delivery of the molecule into the cell by at least 10%.   2. The peptide of claim 1, wherein A is a peptide comprising 5 to about 50 amino acids, and A improves delivery of the molecule into the cell by about 50% to about 100%. .   2. The peptide of claim 1, wherein A is a peptide comprising 5 to about 50 amino acids, and A improves the delivery of the molecule into the cell by about 75% to about 500%. .   The A according to claim 1, characterized in that A is at least 50% similar to any one of SEQ ID NOs: 1 to 68 and A improves the delivery of the molecule into the cell by at least 20%. Said peptide.   The A according to claim 1, characterized in that A is at least 75% similar to any one of SEQ ID NOs: 1 to 68, and A improves delivery of the molecule into the cell by at least 50%. Said peptide.   2. A according to claim 1, characterized in that A is at least 90% similar to any one of SEQ ID NOs: 1 to 68, and A improves delivery of the molecule into the cell by at least 100%. Said peptide.   A is at least 90% in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 67, and SEQ ID NO: 68 2. The peptide of claim 1 that is similar.   The peptide according to claim 1, characterized in that L comprises a sequence of 3 to 50 amino acids and at least 50% of the amino acids in the sequence are neutral.   The peptide according to claim 1, characterized in that L comprises a sequence of 3 to 50 amino acids and at least 60% of the amino acids in the sequence are neutral.   The peptide according to claim 1, characterized in that L comprises a sequence of 3-50 amino acids and at least 75% of the amino acids in the sequence are neutral.   The peptide according to claim 1, characterized in that L comprises a sequence of 3-50 amino acids and at least 30% of the amino acids in the sequence are neutral and polar.   The peptide according to claim 1, characterized in that L comprises a sequence of 6 to 60 amino acids and at least 30% of the amino acids in the sequence are neutral and polar.   The peptide according to claim 1, characterized in that L comprises a sequence of 10 to 25 amino acids and at least 30% of the amino acids in the sequence are neutral and polar. L is the structure

Wherein each X is independently a neutral amino acid;
Each Y is independently a neutral polar amino acid;
m is an integer of 3 to 50,
n is an integer of 1 to 40,
The peptide according to claim 1, wherein when L is not a bond, p is an integer of 1-20.   17. The peptide of claim 16, wherein m is greater than n.   17. The peptide of claim 16, wherein each X is independently glycine, alanine, valine, leucine, or isoleucine.   17. The peptide of claim 16, wherein X is Gly and Y is Ser.   20. The peptide of claim 19, wherein m is 3, n is 1 and p is 3.   The peptide according to claim 1, wherein B is an amino acid sequence having a net positive charge at physiological pH.   The peptide according to claim 1, wherein B is a peptide sequence comprising 5 to 20 positively charged amino acids.   The peptide according to claim 1, wherein B is a positively charged amino acid sequence comprising 5 to 20 Arg residues. B is _Arg 5 _{~Arg 20,} wherein the peptide of claim 1.   A peptide comprising a peptide sequence that is at least 75% similar to the peptides of Table 4, characterized in that it improves the delivery of the molecule into the cell by about 50% to about 100%.   A transfection complex comprising the peptide of claim 1.   27. The transfection complex of claim 26, further comprising a cationic lipid.   28. The transfection complex of claim 27, further comprising a neutral lipid.   A transfection complex comprising a peptide that is at least 75% similar to a peptide sequence selected from the list consisting of SEQ ID NO: 89 to SEQ ID NO: 107, at least one cationic lipid, and at least one helper lipid.   30. The transfection complex of claim 29, further comprising at least one cargo molecule.   31. The transfection complex of claim 30, wherein the cargo molecule is a nucleic acid.   32. The transfection complex of claim 31, wherein the cargo molecule is a DNA or RNA molecule.

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